Method for Angiographically Guided Fine-Needle Diathermy in the Treatment of Corneal Neovascularization

Purpose: To describe a method to assess corneal neovascular (CoNV) complexes and identify feeder vessels for selective arterial fine-needle diathermy (FND). Methods: In patients with CoNV, color photography and corneal indocyanine green angiography (ICGA) and fluorescein angiography are performed. After injection of indocyanine green and sodium fluorescein dye, videography and single-frame images of the region of interest are recorded. Videography is used to measure the time to leakage to assess vessel maturity to guide medical treatment and to discern afferent from efferent vessels. Single-frame images are then selected to locate the number of afferent vessels for surgery, which are selectively cut with a 25-gauge marked needle for the application of FND. Results: Angiography using fluorescein and indocyanine green allows the characterization of CoNV based on assessment of both morphologic (ICGA) and functional (fluorescein angiography) parameters. The time to leakage of fluorescein dye provides important functional information on vessel maturity and helps discern whether medical treatment should be followed before surgical. ICGA allows the identification and delineation of afferent feeder vessels even in the presence of corneal opacities affecting biomicroscopic visibility. Colocalizing the afferent vessel to a visible venous landmark or branch is helpful for placement of the incision and application of FND. Using the described approach, angiographically identified feeder vessels can be selectively treated by FND with minimal thermal energy applied to the corneoscleral limbus. Conclusions: The described method for angiographically guided assessment of CoNV is a useful approach for guiding the medical and surgical treatment of CoNV

Predictive Association of Pre-Operative Defect Areas in the Outer Retinal Layers With Visual Acuity in Macular Hole Surgery

Purpose: The purpose of this study was to develop methods to model the external limiting membrane (ELM) and ellipsoid zone (EZ) within the elevated cuff surrounding a macular hole (MH) to determine if the predicted size of the defect in these layers after virtual flattening was associated with the actual postoperative defect and best corrected visual acuity (BCVA). Methods: Patients were included who had undergone successful MH surgery. The defects in the ELM and EZ after virtual flattening were modeled using in-house software. Main outcomes were postoperative defects in ELM and EZ at 2 months and BCVA at 12 months. Results: Fifty-eight patients were included. BCVA improved from 0.87 (0.31) logMAR pre-operatively to 0.26 (0.21) at 12 months (P < 0.001). For both the ELM and EZ, the predicted virtually flattened pre-operative defects were associated with the actual postoperative defects at 2 months (R-2 = 0.33, P < 0.01 and R-2 = 0.50, P < 0.01, respectively). There was a significant association of BCVA at 12 months (adjusted R-2 = 0.85) with the pre-operative modeled area of the defect in the ELM (P < 0.01) and to a lesser extent with the defect in the EZ (P < 0.01) and base of the MH (P < 0.01). Conclusions: Virtually flattening of the pre-operative defect in the ELM provides important predictive information of visual acuity. Incorporation of tools into commercially available optical coherence tomography (OCT) devices to facilitate such measurements would provide the clinician with important prognostic information. Translational Relevance: We have developed methodology that can potentially be used to predict the postoperative state of the outer retinal layers and the associated visual outcome in patients undergoing surgery for MH

Yield and Viability of Human Limbal Stem Cells From Fresh and Stored Tissue

Purpose: We compared cell number, putative stem cell markers, and clonogenic ability in fresh uncultured human limbal epithelial cells to that obtained from stored organ-cultured tissue. Methods: Cell suspensions were formed from fresh and organ culture–stored human limbal epithelium. Expression of putative stem cell markers ΔNp63 and TrkA was performed using immunofluorescent staining before culture. Colony-forming efficiency (CFE) assays were performed at first passage. The effects of tissue storage, age, and postmortem/culture times were analyzed in a general linear model. Results: Limbal tissue from 94 donors (34 fresh and 60 stored) was compared. Three times more cells were obtained per eye from fresh (35.34 × 104; SD, 17.39) than stored (11.24 × 104; SD, 11.57; P < 0.01) tissue. A higher proportion of cells from fresh tissue were viable (91.9%; SD, 5.7 vs. 85%; SD, 10.8) P < 0.01. Higher total cell expression of ΔNp63 (20.19 × 104; SD, 15.5 vs. 3.28 104; SD, 4.33) and TrkA (59.24 × 104; SD, 13.21 vs. 7.65 × 104; SD, 1.05) was observed in fresh than stored tissue (P < 0.01). Colony-forming efficiency was higher for fresh (1.42; SD, 0.12) than stored (0.43; SD, 0.15; P < 0.01) cells. For stored tissue only, there was a significant inverse relationship between donor age and total number of cells isolated (R2 = 0.27, P < 0.001). Conclusions: Storage of corneoscleral discs in organ culture medium leads to significant reduction in limbal epithelial cell number, expression of ΔNp63 and TrkA, and viability compared to fresh tissue. There is a smaller basal stem cell population in stored compared to fresh tissue

Cross-Country Transportation Efficacy and Clinical Outcomes of Preloaded Large-Diameter Ultra-Thin Descemet Stripping Automated Endothelial Keratoplasty Grafts

PURPOSE: To evaluate the clinical outcomes of preloaded large-diameter ultra-thin grafts for Descemet stripping automated endothelial keratoplasty (UT-DSAEK) after cross-country shipment. METHODS: A laboratory study in an eye bank and a clinical cohort study in an academic tertiary care center were performed. UT-DSAEK (9.5 mm diameter) grafts (n = 7) were prepared, loaded into a commercial device (iGlide; Eurobio, Les Ulis, France), preserved for 4 days at room temperature in transport medium, and analyzed. In a retrospective study, preloaded tissues (n = 39) for clinical use were prepared, transported from Italy to the United Kingdom, and surgically delivered into the eyes of patients undergoing UT-DSAEK. Central and peripheral endothelial cell density (ECD) and viability were measured before and after loading and storage of the grafts in the laboratory study. Clinically, best-corrected visual acuity, ECD before and at final follow-up, dislocation rate, primary graft failure, and surgical time were recorded. RESULTS: In the laboratory study, postcut central graft thickness was 93.3 ± 17.2 μm. ECD and cell mortality did not change significantly before and after preservation (P = 0.8). Cell loss after 4 days of preservation was 1.7% ± 1.6%. Clinically, 39 eyes of 39 patients at final follow-up showed a mean central graft thickness of 88 ± 22 μm and a best-corrected visual acuity of 0.34 ± 0.24 logMAR. Nine of 39 cases (23%) needed rebubbling, and 28% cell loss was observed at final follow-up. CONCLUSIONS: Large-diameter UT-DSAEK grafts can be prepared and preloaded in the eye bank using the iGlide and transported to the surgical center facilitating surgery for patients undergoing UT-DSAEK, potentially reducing tissue wastage, surgical time, and costs related to surgery

Human Conjunctival Stem Cells are Predominantly Located in the Medial Canthal and Inferior Forniceal Areas.

PURPOSE: The conjunctiva plays a key role in ocular surface defence and maintenance of the tear film. Ex vivo expansion of conjunctival epithelial cells offers potential to reconstruct the ocular surface in cases of severe cicatrising disease, but requires initial biopsies rich in stem cells to ensure long-term success. The distribution of human conjunctival stem cells, however, has not been clearly elucidated. METHODS: Whole human cadaveric conjunctiva was retrieved and divided into specific areas for comparison. From each donor, all areas from one specimen were cultured for colony-forming efficiency assays and immunocytochemical studies; all areas from the other specimen were fixed and paraffin embedded for immunohistochemical studies. Expression of CK19, p63, and stem cell markers ABCG2, ΔNp63, and Hsp70 were analyzed. Results were correlated to donor age and postmortem retrieval time. RESULTS: Conjunctiva was retrieved from 13 donors (26 specimens). Colony-forming efficiency and expression of stem cell markers ABCG2, ΔNp63, and Hsp70 in cultures and ABCG2 in fixed tissue were all consistently demonstrated throughout the tissue but with highest levels in the medial canthal and inferior forniceal areas (P < 0.01 for each). Both increasing donor age and longer postmortem retrieval times were associated with significantly lower colony-forming efficiency, stem cell marker expression in cell cultures and ABCG2 expression in fixed tissue. CONCLUSIONS: Biopsies from the medial canthus and inferior forniceal areas, from younger donors, and with short postmortem retrieval times offer the greatest potential to developing conjunctival stem cell-rich epithelial constructs for transplantation

Deformation velocity imaging using optical coherence tomography and its applications to the cornea

Optical coherence tomography (OCT) can monitor human donor corneas non-invasively during the de-swelling process following storage for corneal transplantation, but currently only resultant thickness as a function of time is extracted. To visualize and quantify the mechanism of de-swelling, we present a method exploiting the nanometer sensitivity of the Fourier phase in OCT data to image deformation velocities. The technique was demonstrated by non-invasively showing during de-swelling that osmotic flow through an intact epithelium is negligible and removing the endothelium approximately doubled the initial flow at that interface. The increased functional data further enabled the validation of a mathematical model of the cornea. Included is an efficient method of measuring high temporal resolution (1 minute demonstrated) corneal thickness, using automated collection and semi-automated graph search segmentation. These methods expand OCT capabilities to measure volume change processes for tissues and materials

Association of creatine kinase and skin toxicity in phase I trials of anticancer agents.

BACKGROUND: We investigated the association between skin rash and plasma creatine kinase (CK) levels in oncology phase I trials. METHODS: We analysed data from 295 patients treated at our institution within 25 phase I trials which included CK measurements in the protocol. Trials involved drugs targeting EGFR/HER2, m-TOR, VEGFR, SRC/ABL, aurora kinase, BRAF/MEK, PARP, CDK, A5B1 integrin, as well as oncolytic viruses and vascular disrupting agents. RESULTS: Creatine kinase measurements were available for 278 patients. The highest levels of plasma CK during the trial were seen among patients with Grade (G) 2/3 rash (median 249 U l(-1)) compared with G1 (median 81 U l(-1)) and no rash (median 55 U l(-1)) (P<0.001). There was a significant reduction in CK after the rash resolved (mean 264.2 vs 100.1; P=0.012) in 25 patients, where serial CK values were available. In vitro exposure of human keratinocytes to EGFR, MEK and a PI3Kinase/m-TOR inhibitor led to the increased expression of CK-brain and not CK-muscle or mitochondrial-CK. CONCLUSION: Plasma CK elevation is associated with development of skin rash caused by novel anticancer agents. This should be studied further to characterise different isoforms as this will change the way we report adverse events in oncology phase I clinical trials