Nephronectin (Npnt) is an extracellular matrix (ECM) protein with pleiotropic functions during organogenesis, disease, and homeostasis. Although the ECM plays a crucial role during development and homeostasis of the adult cornea, little is known about the expression of Npnt in the mammalian cornea. Here, we investigated the expression of Npnt during early embryonic and postnatal development, and in adult mouse corneas. We combined ultrastructural and immunohistochemical analyses to study the early formation of the Descemet\'s membrane and how the expression of Npnt relates to key basement membrane proteins. Our section in situ hybridization and immunohistochemical analyses revealed that Npnt mRNA is expressed by the nascent corneal endothelial cells at embryonic day (E) 14.5, whereas the protein is localized in the adjacent extracellular matrix. These expression patterns were maintained in the corneal endothelium and Descemet\'s membrane throughout development and in adult corneas. Ultrastructural analysis revealed discontinuous electron dense regions of protein aggregates at E18.5 that was separated from the endothelial layer by an electron lucent space. At birth (postnatal day, P0), the Descemet\'s membrane was a single layer, which continuously thickened throughout P4, P8, P10, and P14. Npnt was localized to the Descemet\'s membrane by E18.5 and overlapped with Collagens IV and VIII, Laminin, and Perlecan. However, the proteins subsequently shifted and formed distinct layers in the adult cornea, whereby Npnt localized between two Collagen VIII bands and anterior to Collagen IV but overlapped with Laminin and Perlecan. Combined, our results reveal the expression of Npnt in the mouse cornea and define its spatiotemporal localization relative to key basement membrane proteins during the formation of the Descemet\'s membrane and in the adult cornea. Understanding the spatiotemporal expression of Npnt is important for future studies to elucidate its function in the mammalian cornea.

We aimed to determine whether Descemet\'s membrane (DM) scrolling occurs primarily along the vertical or horizontal axis and establish whether oval trephination along the axis of least scrolling can reduce the grade of the scroll.
The longest limbus-to-limbus axis on 28 sclerocorneal discs was taken as the horizontal axis. The horizontal (n = 7) or (right angles to it) vertical (n = 6) axis was marked on DM before peeling it off. The direction and grade of scrolling was observed. Narrow strips (3-4 mm wide) were then cut along the two axes (n = 4 each) and the scrolling pattern was observed. Ellipses (7 × 9 mm) of DM were punched along the two axes (n = 6 each) and the scrolls graded. Immunofluorescent staining for elastin on horizontal and vertical tissue sections from three DM samples was performed. The intensity and thickness of elastin staining were measured.
Twenty-four (85.72%) DM samples showed scrolling along the horizontal axis, none showed scrolling along the vertical axis, and four (14.28%) samples showed a spiral scroll, regardless of which axis was marked (grade 3.7 and 3.6). Vertically oval discs showed significantly reduced scrolling (grade 1.2) compared to horizontally oval discs (grade 3.5). Narrow strips of DM showed a similar scrolling pattern. Immunohistology showed no difference in any of the parameters examined along the two axes or from the center to the periphery.
DM scrolls primarily along the horizontal axis. Vertically oval DM samples show minimal scrolling, which can be an advantage in DMEK. Differential scrolling is not determined by the distribution of elastin.

UNASSIGNED: To report the 21st case showing the rare occurrence of retained Descemet\'s membrane (DM) following penetrating keratoplasty (PKP). We intend to investigate possible etiologies, expected sequelae, and outcome of neodymium-dpoed yttrium alumnium garnet (Nd: YAG) laser membranectomy.
METHODS: Our case is a 74-year-old male who underwent PKP surgery in the right eye secondary to corneal decompensation following cataract surgery in addition to corneal thinning secondary to superficial keratectomy related to the pre-existing climatic droplet keratopathy (CDK). Postoperative assessment revealed a retro-corneal membrane within the anterior chamber, which was affecting his vision.
UNASSIGNED: Based on the post-operative course and the decreased vision as an indication for intervention, it was decided to excise the retained DM. Membranectomy with Nd: YAG laser was performed, and the patient\'s visual acuity measurement improved from 20/400 to 20/25. However, the endothelial cell count decreased from 1479 to 520 cells/mm2 (35 % loss) at 15 months post YAG membranectomy with clear graft. Histopathological examination confirmed the clinical suspicion of a retained DM, since it was absent in the submitted host corneal tissue in addition to the pre-existing CDK.
CONCLUSIONS: Retention of DM following PKP is a rare but possible complication and high index of suspicion is required for proper diagnosis and management to obtain better visual outcome. Nd: YAG laser membranectomy was effective in excising the retained DM and improving vision. Endothelial cell loss following Nd: YAG laser membranectomy as a complication was observed and should be addressed during the treatment plan.

A 62-year-old woman was referred with poor vision following manual small incision cataract surgery. On presentation, the uncorrected distance visual acuity in the involved eye was 3/60, whereas slit-lamp examination revealed a central corneal edema with the peripheral cornea relatively clear. Direct focal examination with a narrow slit upper border and lower margin of detached rolled up Descemet\'s membrane (DM) could be visualized. We performed a novel surgical approach, \"double-bubble pneumo-descemetopexy.\" The surgical procedure included unrolling of DM with \"small air bubble\" and descemetopexy with \"big bubble.\" No postoperative complications were observed, and best corrected distance visual acuity improved to 6/9 at 6 weeks. The patient had a clear cornea and maintained BCVA 6/9 during 18 months at follow-up. Double-bubble pneumo-descemetopexy, a more controlled technique, provides a satisfactory anatomical and visual outcome in DMD without the need for endothelial keratoplasty (Descemet\'s stripping endothelial keratoplasty or DMEK) or penetrating keratoplasty.

Fuchs\' endothelial corneal dystrophy (FECD) is progressive corneal endothelium dysfunction, characterised by corneal oedema, and potential blindness if left untreated. Keratoplasty is the only definitive treatment to restore vision in FECD, with different surgical techniques being described. The corneal transplant has been described as the most commonly performed and most successful allogenic transplant globally; therefore, it is crucial to dissect it further since a large proportion of the population worldwide is likely to be impacted. We feel that an updated literature review is both very relevant and necessary at present and aim to amalgamate more recent data on the topic (including meta-analyses, systematic reviews, and randomised control trials (RCTs), among others). We acknowledge that the paucity of reliable data limits progress for FECD and that there are existing ethical complexities in performing prospective trials on patients.  Traditionally, the surgery for FECD was limited to penetrating keratoplasty (PK), yet recent developments have introduced more advanced procedures and adapted the existing ones, to provide treatment specific to the disease-affected corneal layers. The questions we will address encompass: how does the severity of FECD govern the treatment options available, what are the differences between PK and types of endothelial keratoplasty (EK), what are the expected clinical outcomes of each of these operations, what are the potential concerns with the idealistic descemetorhexis surgery, and what do we envisage for times to come? Besides this, novel minimally-invasive pharmacological techniques are now being trialled, such as Rho kinase (ROCK) inhibition and cultured endothelial cells (CECs), which may drastically improve the dependence on corneal donors. We examine and critically appraise the literature to explore the understanding of FECD, and the treatment options that exist: historically, currently, and those anticipated for the future.

Descemet\'s membrane (DM), the basement membrane of the corneal endothelium, is formed from the extracellular matrix (ECM) secreted by corneal endothelial cells. The ECM supports the growth and function of the corneal endothelial cells. Changes to DM are central to the diagnosis of the most common corneal endothelial disease, Fuchs endothelial corneal dystrophy (FECD). Changes in DM are also noted in systemic diseases such as diabetes mellitus. In FECD, the DM progressively accumulates guttae, \"drop-like deposits\" that disrupt the corneal endothelial cell monolayer. While the pathophysiologic changes to corneal endothelial cells in the course of FECD have been well described and reviewed, the changes to DM have received limited attention. The reciprocity of influence between the corneal endothelial cells and DM demands full attention to the latter in our search for novel treatment and preventive strategies. In this review, we discuss what is known about the formation and composition of DM and how it changes in FECD and other conditions. We review characteristics of guttae and the interplay between corneal endothelial cells and guttae, particularly as it might apply to future cell-based and genetic therapies for FECD.

To explore the use of a thermoreversible copolymer gel coating to prevent donor tissue scrolling in Descemet\'s membrane endothelial keratoplasty (DMEK).
PLGA-PEG-PLGA triblock copolymer was synthesised via ring opening polymerisation. Two formulations were fabricated and gelation properties characterised using rheological analyses. Endothelial cytotoxicity of the copolymer was assessed using a Trypan Blue exclusion assay. Thickness of the copolymer gel coating on the endothelial surface was analysed using anterior segment optical coherence tomography (OCT) (RTVue-100, Optovue Inc.). Gold nanoparticles were added to the copolymer to aid visualisation using OCT. Prevention of Descemet membrane donor scrolling was represented via a novel, in vitro, immersion of copolymer coated donor graft material.
Two different formulations of PLGA-PEG-PLGA copolymer were successfully fabricated and the desired peak gelling temperature of 24°C was achieved by polymer blending. Application of 20%, 30% and 40% (wt/vol) polymer concentrations resulted in a statistically significant increase in polymer thickness on the endothelium (p < 0.001). There was no detectable endothelial cytotoxicity. The polymer was easy to apply to the endothelium and prevented scrolling of the DMEK graft.
This PLGA-PEG-PLGA thermoreversible copolymer gel could be exploited as a therapeutic aid for preventing DMEK graft scrolling.

To compare posterior corneal morphology between older treated and younger untreated children with primary congenital glaucoma (PCG) using anterior segment optical coherence tomography (ASOCT) and intraoperative OCT (iOCT), respectively.
In this comparative study, ASOCT of older PCG children were compared with iOCT of younger untreated PCG patients. Differences between the two groups with respect to posterior corneal morphology were studied.
Observed morphological patterns within posterior cornea in older treated (age: 72-300 months) children (87 eyes) included Descemet\'s membrane (DM) excrescences (70%), thickened DM (35%), intracameral twin protuberances (92%), and DM detachment (26%). Changes within pre-Descemet\'s layer (PDL) (28%) included thickening, breaks, and detachments. Extent of Haab\'s striae was associated with thickness of DM/PDL complex (P = 0.008) when analyzed in the treated group. In contrast, in the untreated group (n = 53 eyes, age 1-63 months), posterior corneal changes were limited to diffuse hyper-reflectivity of the DM/PDL complex, with absence of DM tears.
Posterior cornea thickens and Haab\'s striae become more circumscribed in eyes of older treated children compared to untreated PCG eyes, probably reflecting a healing response of posterior cornea over time.

The purpose of this study was to examine the effect of topical and/or oral angiotensin converting enzyme II inhibitor and TGF-beta signaling blocker losartan on corneal stromal fibrosis that developed in rabbit corneas after Descemetorhexis removal of central Descemet\'s membrane and corneal endothelium. Twenty-eight New Zealand white rabbits were included and either had 8 mm central Descemetorhexis or sham control surgery without Descemetorhexis in one eye. Groups of 4 eyes without Descemetorhexis were treated for one month with no medications, topical losartan or oral losartan. Groups of 4 eyes with Descemetorhexis were treated with topical and oral vehicle, topical losartan, oral losartan, or both topical losartan and oral losartan for one month. Standardized slit lamp photos were obtained with central opacity intensity measured with ImageJ. The posterior fibrotic zone of corneas was measured on immunohistochemistry for alpha-smooth muscle actin (SMA) and keratocan using QuPath analysis. Collagen type IV expression in the posterior cornea was quantitated with ImageJ and duplex immunohistochemistry for collagen type IV and TGF beta-1. After Descemetorhexis, topical, but not oral, losartan decreased the intensity of central stromal opacity, reduced peripheral corneal scarring, and decreased alpha-smooth muscle actin myofibroblast fibrosis area compared to corneas that had Descemetorhexis and treatment with vehicles alone. Topical losartan decreased posterior stromal cellular, non-Descemet\'s membrane, collagen type IV production, that is likely stimulated by TGF beta as part of a negative regulatory feedback mechanism, compared to vehicle treatment at one month after Descemetorhexis. Topical losartan is likely to be effective in reducing corneal scarring fibrosis produced by traumatic injury, microbial infection, and some corneal diseases and surgeries.

The purpose of this investigation was to study Descemet\'s membrane and corneal endothelial regeneration, myofibroblast generation and disappearance, and TGF beta-1 localization after Descemet\'s membrane-endothelial excision (Descemetorhexis) in rabbits. Thirty-six rabbits had 8 mm Descemetorhexis and standardized slit lamp photos at 1, 2 and 4 days, 1, 2 and 4 weeks, and 2, 4 and 6 months, as well as multiplex IHC for stromal cell markers keratocan, vimentin, and alpha-smooth muscle actin (SMA); basement membrane (BM) components perlecan, nidogen-1, laminin alpha-5, and collagen type IV; and corneal endothelial marker Na,K-ATPase β1, and TGF beta-1, with ImageJ quantitation. Stromal transparency increased from the periphery beginning at two months after injury and progressed into the central cornea by six months. At six months, central transparency was primarily limited by persistent mid-stromal neovascularization. Stromal myofibroblast zone thickness in the posterior stroma peaked at one month after injury, and then progressively decreased until to six months when few myofibroblasts remained. The regeneration of a laminin alpha-5 and nidogen-1 Descemet\'s membrane \"railroad track\" structure was accompanied by corneal endothelial closure and stromal cell production of BM components in corneas from four to six months after injury. TGF beta-1 deposition at the posterior corneal surface from the aqueous humor peaked at one day after Descemetorhexis and diminished even before regeneration of the endothelium and Descemet\'s membrane. This decrease was associated with collagen type IV protein production by corneal fibroblasts, and possibly myofibroblasts, in the posterior stroma. Descemet\'s membrane and the corneal endothelium regenerated in the rabbit cornea by six months after eight mm Descemetorhexis. Real-time quantitative RT-PCR experiments in vitro with marker-verified rabbit corneal cells found that 5 ng/ml or 10 ng/ml TGF beta-1 upregulated col4a1 or col4a2 mRNA expression after 6 h or 12 h of exposure in corneal fibroblasts, but not in myofibroblasts. Stromal cells produced large amounts of collagen type IV that likely decreased TGF beta-1 penetration into the stroma and facilitated the resolution of myofibroblast-generated fibrosis.