Morphological characterization and clinical effects of stromal alterations after intracorneal ring segment implantation in keratoconus

Purpose To analyze the histological and (ultra)structural stromal tissue changes after femtosecond (Fs) laser–assisted intra corneal ring segment (ICRS) implantation and their refractive and topographic efects in patients with keratoconus. Methods This monocentric retrospective case series included 15 consecutive patients with clinical peri-segmental lamellar channel deposits after treatment with Fs-ICRS implantation for keratoconus. The stromal changes were investigated using in vivo confocal microscopy. Two patients underwent a penetrating keratoplasty after the Fs-ICRS implantation; the explanted corneas were processed for histopathology and transmission electron microscopy (TEM). Refractive and topographic efects were investigated comparing the uncorrected (UDVA) and corrected (CDVA) distance visual acuity, spherical equivalent (SE), fat (K1), steep (K2), and steepest (Kmax) keratometry before and after detection of lamellar channel deposits. Results In vivo confocal microscopy revealed difuse linear and focal granular hyperrefective structures. Histologically, there was mild proliferation of fbroblasts and fbrosis. TEM demonstrated focal accumulations of degenerated keratocytes with cytoplasmic lipid inclusions. There were no signifcant changes for UDVA (Δ=0.0±0.2 logMAR; p=0.67), CDVA (Δ=0.0±0.1 logMAR; p=0.32), SE (Δ 0.1±0.9 D; p=0.22), K1 (Δ=0.3±1.0 D; p=0.28), K2 (Δ=0.1±0.9 D; p=0.51), and Kmax (Δ=0.3±1.5 D; p=0.17). Conclusions Two types of structural stromal changes were identifed: (1) difuse peri-segmental fbrosis and (2) lamellar channel deposits. These structural changes showed no evidence of a relevant refractive or topographic efect

Corneal epithelial ingrowth after perforating corneal injury: a case report

Background Epithelial ingrowth is a rare complication after ocular perforation and can become manifest many years after the primary trauma. Case presentation A 49-year-old patient presented with a positive Seidel test of unclear origin at her left eye, as well as a sharply defned anterior-stromal corneal scar at both eyes. Prior operations included a bilateral laser-assisted blepharoplasty 3 months earlier. The patient indicated to have been on holiday to France 5 months earlier, during an ongoing oak processionary moth caterpillars infestation. The examination using confocal microscopy confrmed a corneal perforation at the left eye and revealed corneal epithelial ingrowth capped with scarred stroma in both eyes. We performed a penetrating keratoplasty at the left eye. The scarred and perforated host cornea was divided into 4 pieces for further investigation: microbiology (negative), virology (negative), histology and transmission electron microscopy (TEM). Histology revealed diferently structured epithelium, centrally inverted into the stroma through defects in Bowman’s layer. TEM revealed full thickness corneal perforation with an epithelial plug extending to the lower third of the cornea, but without evidence of epithelial cell migration into the anterior chamber. Our diferential diagnosis of the unclear positive Seidel test with epithelial ingrowth was as follows: (1) corneal perfo‑ ration by hairs of the oak processionary moth caterpillar, although no hairs could be found histologically; (2) corneal perforation during laser-assisted blepharoplasty, which may be supported by the presence of pigmented cells on the posterior surface of Descemet´s membrane, pointing to a possible iris injury. Conclusion Consequently, we highlighted that contact lenses can be useful, safe and inexpensive protective devices in upper eyelid procedures to protect the cornea against mechanical iatrogenic trauma

Recurrence of paraproteinemic keratopathy after penetrating keratoplasty and its assessment with confocal microscopy

Purpose To report on a case of recurrence of paraproteinemic keratopathy (PPK) associated with monoclonal gammopathy after bilateral penetrating keratoplasty. Observations Penetrating keratoplasty was performed on both eyes of a 45-year-old man due to bilateral progressive corneal stromal clouding. Recurrence of the corneal stromal opacities accompanied by a decrease in visual acuity was observed on slit-lamp examination already two years after penetrating keratoplasty. Confocal laser scanning microscopy (CLSM) of the corneal grafts performed three years after penetrating keratoplasty showed bilateral morphological changes identical to that found in the patient's corneas prior to penetrating keratoplasty. A hematological work-up revealed monoclonal gammopathy of type IgG kappa. The histochemical examination of the explanted corneas confirmed the diagnosis of PPK. Conclusions and importance Paraproteinemic keratopathy is an underdiagnosed ophthalmological condition, which may be associated with potentially life-threatening hematologic disorders. A hematological workup should be performed in patients with corneal opacities of uncertain etiology. Penetrating keratoplasty should be performed with caution in patients with monoclonal gammopathy due to the possibility of a very fast recurrence of PPK in the corneal graft. This is the first presentation of the recurrence of flake like PPK after penetrating keratoplasty assessed with CLSM

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

Isolation and enrichment of melanocytes from human corneal limbus using CD117 (c-Kit) as selection marker

Abstract Limbal melanocytes (LM) are located in the basal epithelial layer of the corneoscleral limbus and interact with adjacent limbal epithelial progenitor cells. The exploration of their biological role in the maintenance of the limbal stem cell niche has been limited by the difficulty of LM isolation and cultivation. Here, we report on a facile protocol for the efficient isolation and enrichment of pure populations of human LMs by fluorescence-activated cell sorting (FACS) using antibodies raised against the cell surface marker CD117 (c-Kit). The enriched LMs retain self-renewal capacity and sustained melanin production, and are suitable to study the potential of LMs in stem cell-based corneal tissue engineering

Posttranslational modification and mutation of histidine 50 trigger alpha synuclein aggregation and toxicity

Background Aggregation and aggregation-mediated formation of toxic alpha synuclein (aSyn) species have been linked to the pathogenesis of sporadic and monogenic Parkinson’s disease (PD). A novel H50Q mutation of aSyn, resulting in the substitution of histidine by glutamine, has recently been identified in PD patients. We have previously shown that the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) induces the formation of HNE-aSyn adducts, thereby promoting aSyn oligomerization and increasing its extracellular toxicity to human dopaminergic neurons. Intriguingly, we identified histidine 50 (H50) of aSyn as one of the HNE modification target residues. These converging lines of evidence support the hypothesis that changes in H50 via posttranslational modification (PTM) and mutation trigger the formation of aggregated, toxic aSyn species, which interfere with cellular homeostasis. In the present study, we aim to elucidate 1) the role of H50 in HNE-mediated aSyn aggregation and toxicity, and 2) the impact of H50 mutation on aSyn pathology. Besides the PD-related H50Q, we analyze a PD-unrelated control mutation, in which H50 is replaced by an arginine residue (H50R). Results Analysis of HNE-treated aSyn revealed that H50 is the most susceptible residue of aSyn to HNE modification and is crucial for HNE-mediated aSyn oligomerization. Overexpression of aSyn with substituted H50 in H4 neuroglioma cells reduced HNE-induced cell damage, indicating a pivotal role of H50 in HNE modification-induced aSyn toxicity. Furthermore, we showed in vitro that H50Q/R mutations substantially increase the formation of high density and fibrillar aSyn species, and potentiate the oligomerization propensity of aSyn in the presence of a nitrating agent. Cell-based experiments also revealed that overexpression of H50Q aSyn in H4 cells promotes aSyn oligomerization. Importantly, overexpression of both H50Q/R aSyn mutants in H4 cells significantly increased cell death when compared to wild type aSyn. This increase in cell death was further exacerbated by the application of H2O2. Conclusion A dual approach addressing alterations of H50 showed that either H50 PTM or mutation trigger aSyn aggregation and toxicity, suggesting an important role of aSyn H50 in the pathogenesis of both sporadic and monogenic PD