Characterisations of Pre-Descemet's (Dua's) layer for its clinical application in keratoplasty

There exists a newly discovered, well defined, acellular, strong layer, termed pre-Descemets layer or Dua’s layer (PDL), in the cornea just anterior to the Descemets membrane. This, with the Descemets membrane, separates along the last row of keratocytes in most cases of deep anterior lamellar keratoplasty with the big bubble technique. Recognition of this layer has considerable impact on lamellar corneal surgery, understanding of posterior corneal biomechanics and posterior corneal pathology, such as descemetocele, acute hydrops and pre-Descemets dystrophies. The aim of this work was to understand the dynamics of big bubble formation in the context of the known architecture of the cornea stroma, ascertain how type 1 (air between deep stroma and PDL), type 2 (air between PDL and Descemets membrane) and mixed bubbles (combination of type 1 and type 2) form and measure the pressure and volume of air required to produce big bubbles in vitro, including the intra-bubble pressure and volume for the different types of big bubbles. We also aimed to characterise the optical coherence tomography characteristics of the different layers in the wall of the big bubbles to help surgeons identify bubbles and understand the structures seen by intra-operative OCT. Finally we evaluated the endothelial cell density and viability in tissue samples obtained for Descemets membrane endothelial keratoplasty (DMEK) and pre-Descemets endothelial keratoplasty (PDEK) by the pneumodissection technique. Air was injected in 145 corneo-scleral samples, which were unsuitable for transplantation. Samples were obtained in organ culture medium from the UK eye banks and transferred to balanced salt solution ready for injection. Different types of big bubble formed were ascertained. Air pressure and volume required to create the big bubble in simulated deep anterior lamellar keratoplasty were measured. It was found that PDL could withstand a high pressure before bursting at around 700 mm of Hg. Accurate measurements of type-2 big bubble proved challenging. The volume of the type-1 BB was fairly consistent at 0.1ml. The movement of air injected in the corneal stroma was studied from the point of exit from the needle tip to complete aeration of the stroma and formation of a BB. This was video recorded and analysed. A very consistent pattern of air movement was observed. The initial movement was predominantly radial from the needle tip to the limbus, then circular in a clock-wise and counter clock-wise direction circumferentially along the limbus, then centripetally to fill the stroma. All type 1 BB started in the centre as multiple small bubbles which coalesced to form a BB. Almost all type 2 BB started at the periphery near the limbus. Ultrastructural examination of the point of commencement of type 2 BB revealed the presence of clusters of fenestrations, which most likely allow air to escape from the otherwise impervious PDL to access the plane between PDL and DM. This was a novel discovery and explained how type 2 BB formed and why they almost always start at the periphery. The consistent pattern of passage of air was in concordance with the known microarchitecture of the central and peripheral corneal stroma. Optical coherence tomography (OCT) characteristics of different types of big bubbles were studied. Samples obtained from the UK eye banks were scanned with Fourier-domain (FD-OCT), while that obtained from Canada eye bank were scanned with Time-domain (TD-OCT). A special clamp was used to affix the corneo-scleral sample on the OCT table with its posterior surface face the machine and mounted on artificial anterior chamber. It was found that FD-OCT could demonstrate type 1 BB wall as two parallel, double contour, hyper-reflective lines with hypo-reflective space in between. It also revealed that in type-2 BB, the posterior wall showed a parallel, double-contour curved hyper-reflective line with a dark space in between. This probably corresponds to the banded and non-banded zones of DM. Dua’s layer presents as a single hyper-reflective line. In TD-OCT, the posterior wall of type-1 and type-2 BB showed a single hyper-reflective curved line rather than the double-contour line. This finding will help cornea surgeons to identify and interpret different layers of big bubble intra-operatively with high resolution OCT devices. Endothelial cell density of PDEK and DMEK tissue were calculated. Endothelial cells were counted using light microscope before pneumodissection. Air was then injected to ascertain the creation of type-1 and type-2 BB. Tissue was then harvested by trephination and endothelial cell density of both types were calculated again. It was found that the corneal endothelial cell count in PEDK tissue preparation is no worse, if not slightly better than, in DMEK tissue prepared by pneumodissection. Therefore, PDEK preparation represents a viable graft preparation technique.