Design and validation of a custom-made system to measure transepithelial electrical impedance in human corneas preserved in active storage machine.

Corneal epithelial barrier represents one of the major limitations to ocular drug delivery and can be explored non-invasively through the evaluation of its electrical properties. Human corneas stored in active storage machine (ASM) could represent an interesting physiological model to explore transcorneal drug penetration. We designed a new system adapted to human corneas preserved in ASM to explore corneal epithelial barrier function ex-vivo. A bipolar set-up including Ag/AgCl electrodes adaptors to fit the corneal ASM and a dedicated software was designed and tested on freshly excised porcine corneas (n = 59) and human corneas stored 14 days in ASM (n = 6). Porcine corneas presented significant and proportional decrease in corneal impedance in response to increasing-size epithelial ulcerations and acute exposure to benzalkonium chloride (BAC) 0.01 and 0.05%. Human corneas stored 14 days in ASM presented a significant increase in corneal impedance associated with the restoration of a multi-layer epithelium and an enhanced expression of tight junctions markers zonula occludens 1, claudin 1 and occludin. These results support the relevance of the developed approach to pursue the exploration and development of human corneas stored in ASM as a physiological pharmacological model.

Technique of flat-mount immunostaining for mapping the olfactory epithelium and counting the olfactory sensory neurons.

The pathophysiology underlying olfactory dysfunction is still poorly understood, and more efficient biomolecular tools are necessary to explore this aspect. Immunohistochemistry (IHC) on cross sections is one of the major tools to study the olfactory epithelium (OE), but does not allow reliable counting of olfactory sensory neurons (OSNs) or cartography of the OE. In this study, we want to present an easy immunostaining technique to compensate for these defects of IHC. Using the rat model, we first validated and pre-screened the key OSN markers by IHC on cross sections of the OE. Tuj-1, OMP, DCX, PGP9.5, and N-cadherin were selected for immunostaining on flat-mounted OE because of their staining of OSN dendrites. A simple technique for immunostaining on flat-mounted septal OE was developed: fixation of the isolated septum mucosa in 0.5% paraformaldehyde (PFA) preceded by pretreatment of the rat head in 1% PFA for 1 hour. This technique allowed us to correctly reveal the olfactory areas using all the 5 selected markers on septum mucosa. By combining the mature OSN marker (OMP) and an immature OSN marker (Tuj-1), we quantified the mature (OMP+, Tuj-1-), immature (OMP-, Tuj-1+), transitory (OMP+, Tuj-1+) and total OSN density on septal OE. They were respectively 42080 ± 11820, 49384 ± 7134, 14448 ± 5865 and 105912 ± 13899 cells per mm2 (mean ± SD). Finally, the same immunostaining technique described above was performed with Tuj-1 for OE cartography on ethmoid turbinates without flat-mount.

Dropless penetrating keratoplasty using a subconjunctival dexamethasone implant: safety pilot study.

BACKGROUND/AIM: Rejection is the main cause of graft failure after penetrating keratoplasty (PK). Its prevention by repeated instillation of steroid eye-drops has not evolved in decades. Poor adherence and discontinuous nature of eye-drop treatment may explain some PK failures. In a rabbit model, we previously demonstrated that a subconjunctival dexamethasone implant was well tolerated and prevented rejection efficiently in the first 5-6 weeks. This clinical trial investigates its tolerance and safety after PK. METHODS: Single-centre, phase II non-randomised tolerance and safety pilot study (NCT02834260). Designed to analyse the risk of elevated intraocular pressure (IOP), discomfort and resorption time. Fourteen patients with a low rejection risk indication of PK were enrolled between January 2017 and August 2018. The implant was injected in the 12 o'clock position, 5 mm from the limbus, at the end of PK. A steroid eye-drop treatment was planned when implant resorption was complete. Patients were monitored regularly for 12 months: IOP (main outcome measure at 1 month), discomfort and redness scores, implant status, rejection episode and central corneal thickness by optical coherence tomography. An independent data safety monitoring committee verified safety aspects. RESULTS: No increase in IOP or other adverse event related to the implant was observed. Average resorption time was 6 weeks. The switch to steroid eye-drops was uneventful. One patient, included despite preoperative corneal neovascularisation (unintended protocol deviation) experienced a rejection. CONCLUSIONS: This is the first proof of concept that dropless immunosuppression is possible after low rejection risk PK. TRIAL REGISTRATION NUMBER: NCT02834260.

Objective measurement of adherence to topical steroid medication after penetrating keratoplasty using an electronic monitoring aid: A pilot study.

Objectives: Corneal transplantation is the most common transplant worldwide and its success critically depends on the management of corneal graft rejection through topical steroid therapy during the first 12 months after surgery. There is currently no published data on adherence after keratoplasty. This pilot study aims to explore the adherence to topical steroid after penetrating keratoplasty using a smart electronic device. Methods: Thirty patients undergoing penetrating keratoplasty were included to evaluate the adherence to topical dexamethasone medication for 12 months after surgery. Patients received the usual post-transplantation treatment (topical dexamethasone) and follow-up after surgery (day 15, months 1, 2, 3, 4, 5, 6, 9, and 12). Adherence to treatment was monitored using the KaliJAR device (Kali Care, Santa Clara, CA, USA), which recorded the number of single-dose units (SDU) discarded. At control visits, data recorded by the device were compared to the manually count of SDU. Adherence ratio and individual adherence curve were explored for all patients. Results: Data from 27 patients showed a high agreement between adherence ratio calculated based on the device data and obtained from manual counting of the discarded SDU (intraclass coefficient correlation of 0.87 [95% CI: 0.738-0.938]). Mean adherence to the treatment over the 12-month study period was 95.2 ± 4%. Conclusions: Adherence to topical dexamethasone for 12 months after corneal transplantation was high. The connected device was able to record accurately the discarded SDU. This approach would be a particular interest in the early identification and personalized follow-up of poorly adherent patients.

Exploration of the ocular surface infection by SARS-CoV-2 and implications for corneal donation: An ex vivo study.

BACKGROUND: The risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission through corneal graft is an ongoing debate and leads to strict restrictions in corneas procurement, leading to a major decrease in eye banking activity. The aims of this study are to specifically assess the capacity of human cornea to be infected by SARS-CoV-2 and promote its replication ex vivo, and to evaluate the real-life risk of corneal contamination by detecting SARS-CoV-2 RNA in corneas retrieved in donors diagnosed with Coronavirus Disease 2019 (COVID-19) and nonaffected donors. METHODS AND FINDINGS: To assess the capacity of human cornea to be infected by SARS-CoV-2, the expression pattern of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE-2) and activators TMPRSS2 and Cathepsins B and L in ocular surface tissues from nonaffected donors was explored by immunohistochemistry (n = 10 corneas, 78 ± 11 years, 40% female) and qPCR (n = 5 corneas, 80 ± 12 years, 40% female). Additionally, 5 freshly excised corneas (80 ± 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (106 median tissue culture infectious dose (TCID50)/mL). Viral RNA was extracted from tissues and culture media and quantified by reverse transcription quantitative PCR (RT-qPCR) (viral RNA copies) 30 minutes (H0) and 24 hours (H24) after infection. To assess the risk of corneal contamination by SARS-CoV-2, viral RNA was tested by RT-qPCR (Ct value) in both corneas and organ culture media from 14 donors diagnosed with COVID-19 (74 ± 10 years, 29% female) and 26 healthy donors (79 ± 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 ± 13 years, 29% female). The expression of receptor and activators was variable among samples at both protein and mRNA level. Based on immunohistochemistry findings, ACE-2 was localized mainly in the most superficial epithelial cells of peripheral cornea, limbus, and conjunctiva, whereas TMPRSS2 was mostly expressed in all layers of bulbar conjunctiva. A significant increase in total and positive strands of IP4 RNA sequence (RdRp viral gene) was observed from 30 minutes to 24 hours postinfection in central cornea (1.1 × 108 [95% CI: 6.4 × 107 to 2.4 × 108] to 3.0 × 109 [1.4 × 109 to 5.3 × 109], p = 0.0039 and 2.2 × 107 [1.4 × 107 to 3.6 × 107] to 5.1 × 107 [2.9 × 107 to 7.5 × 107], p = 0.0117, respectively) and in corneoscleral rim (4.5 × 109 [2.7 × 109 to 9.6 × 109] to 3.9 × 1010 [2.6 × 1010 to 4.4 × 1010], p = 0.0039 and 3.1 × 108 [1.2 × 108 to 5.3 × 108] to 7.8 × 108 [3.9 × 108 to 9.9 × 108], p = 0.0391, respectively). Viral RNA copies in ex vivo corneas were highly variable from one donor to another. Finally, viral RNA was detected in 3 out of 28 corneas (11%) from donors diagnosed with COVID-19. All samples from the 159 nonaffected donors were negative for SARS-CoV-2 RNA. The main limitation of this study relates to the limited sample size, due to limited access to donors diagnosed with COVID-19 and concomitant decrease in the procurement corneas from nonaffected donors. CONCLUSIONS: In this study, we observed the expression of SARS-CoV-2 receptors and activators at the human ocular surface and a variable increase in viral RNA copies 24 hours after experimental infection of freshly excised human corneas. We also found viral RNA only in a very limited percentage of donors with positive nasopharyngeal PCR. The low rate of positivity in donors diagnosed with COVID-19 calls into question the utility of donor selection algorithms. TRIAL REGISTRATION: Agence de la Biomédecine, PFS-20-011 https://www.agence-biomedecine.fr/.

Tissue engineered endothelial keratoplasty in rabbit: tips and tricks.

PURPOSE: To report a detailed surgical procedure of tissue engineered endothelial keratoplasty (TEEK) in a rabbit model and its postoperative evaluation. METHODS: TEEKs were prepared 7 days before transplantation by seeding human or rabbit corneal endothelial cells on either femtosecond laser-cut ultrathin human stromal lamellae (fs-UTSL) or femtosecond laser-cut human anterior lens capsule (fs-HALC). Thirty transplantations were performed on aphakic eyes. Recombinant tissue plasminogen activator (rTPA) was used throughout the surgery. The native endothelium was removed by full-surface scraping and central descemetorhexis. The transplantation was performed as a human Descemet's membrane endothelial keratoplasty. Controls included Descemetorhexis only and transplantation of carrier alone. Postoperative follow-up was performed by slit lamp and optical coherence tomography, followed by histology. RESULTS: Controls remained oedematous. No fibrin occurred during surgery. All but three TEEKs adhered immediately. One/6 fs-UTSL and 9/16 fs-HALC cleared perfectly (p = 0.161). All failures could be explained by at least one of the following causes intraoperative bleeding, vitreous prolapsus, early partial detachment, postoperative irido corneal synechiea/angle closure. Presumed immune rejection was observed in three rabbits only after 4 weeks. Immunostaining with anti-human CD166 allowed to perfectly differentiate human cells from rabbit cells. In successful TEEK at 3 or 4 weeks, human cells formed a normal endothelium and started migrating outside the carrier. CONCLUSION: Though the transplantation of a TEEK in rabbits is a complex model with many causes of failure, established procedure including use of rTPA allows reliable preclinical study. In addition, we suggest that fs-HALC might be a potential carrier for TEEK.

Inter-individual differences in foveal shape in a scavenging raptor, the black kite Milvus migrans.

Birds, and especially raptors, are believed to forage mainly using visual cues. Indeed, raptors (scavengers and predators) have the highest visual acuity known to date. However, scavengers and predators differ in their visual systems such as in their foveal configuration. While the function of the foveal shape remains unknown, individual variation has never been quantified in birds. In this study, we examined whether foveal shape differs among individuals in relation to eye size, sex, age, eye (left or right) and genetic proximity in a scavenging raptor, the black kite Milvus migrans. We assessed foveal shape in 47 individuals using spectral domain optical coherence tomography (OCT) and geometric morphometric analysis. We found that foveal depth was significantly related to eye size. While foveal width also increased with eye size, it was strongly related to age; younger individuals had a wider fovea with a more pronounced rim. We found no relationship between foveal shape and genetic proximity, suggesting that foveal shape is not a hereditary trait. Our study revealed that the shape of the fovea is directly linked to eye size and that the physical structure of the fovea may develop during the entire life of black kites.

Retinal topography in two species of flamingo (Phoenicopteriformes: Phoenicopteridae).

In this study, we assessed eye morphology and retinal topography in two flamingo species, the Caribbean flamingo (Phoenicopterus ruber) and the Chilean flamingo (P. chilensis). Eye morphology is similar in both species and cornea size relative to eye size (C:A ratio) is intermediate between those previously reported for diurnal and nocturnal birds. Using stereology and retinal whole mounts, we estimate that the total number of Nissl-stained neurons in the retinal ganglion cell (RGC) layer in the Caribbean and Chilean flamingo is ~1.70 and 1.38 million, respectively. Both species have a well-defined visual streak with a peak neuron density of between 13,000 and 16,000 cells mm-2 located in a small central area. Neurons in the high-density regions are smaller and more homogeneous compared to those in medium- and low-density regions. Peak anatomical spatial resolving power in both species is approximately 10-11 cycles/deg. En-face images of the fundus in live Caribbean flamingos acquired using spectral domain optical coherence tomography (SD-OCT) revealed a thin, dark band running nasotemporally just dorsal to the pecten, which aligned with the visual streak in the retinal topography maps. Cross-sectional images (B-scans) obtained with SD-OCT showed that this dark band corresponds with an area of retinal thickening compared to adjacent areas. Neither the retinal whole mounts, nor the SD-OCT imaging revealed any evidence of a central fovea in either species. Overall, we suggest that eye morphology and retinal topography in flamingos reflects their cathemeral activity pattern and the physical nature of the habitats in which they live.

Chronic Intermittent Hypoxia Alters Rat Ophthalmic Artery Reactivity Through Oxidative Stress, Endothelin and Endothelium-Derived Hyperpolarizing Pathways.

Purpose: Obstructive sleep apnea recently has been associated with a higher frequency of ischemic optic neuropathies. Intermittent hypoxia (IH) has been proposed as a major component of obstructive sleep apnea cardiovascular consequences. However, there currently are no pathophysiologic data regarding the effect of IH on the ocular vascular system. Thus, we assessed the impact of chronic IH exposure on the morphology and vascular reactivity of the rat ophthalmic artery (OA). Methods: Rats were exposed to 14 days of IH or normoxia (NX). Ophthalmic artery reactivity was studied using wire myography in rats treated or not with tempol (1 mM/day). Expression of endothelin-1 (ET-1) and its receptors, and of the three nitric oxide synthase (NOS) isoform genes was quantified using quantitative polymerase chain reaction (qPCR) in the retina and optic nerve. Structural alterations (optical and electron microscopy) and superoxide anion production were studied in OA sections. Results: Superoxide ion expression in the OA wall was increased by 23% after IH exposure. Ophthalmic artery contractile response to 3.10-8 M ET-1 was increased by 18.6% and nitric oxide-mediated relaxation was significantly delayed in IH compared to NX rats. In the absence of nitric oxide, cytochrome P450 blockade increased relaxation to acetylcholine in IH rats and delayed it in NX rats. Tempol treatment abolished the IH-induced changes in OA reactivity. Conclusions: These results strongly suggest that chronic IH induces oxidative stress in the rat OA, associated with endothelial dysfunction through alterations of nitric oxide and endothelium-derived hyperpolarising factors (EDHF) pathways.

High intensity focused ultrasound cyclocoagulation in dogs with primary glaucoma: a preliminary study.

The objective was to assess the effect of high intensity focused ultrasound (HIFU) on intraocular pressure (IOP) in dogs with primary glaucoma (PG). Seven dogs (13 eyes) presenting with PG as diagnosed by a raised IOP (> 20 mm Hg) associated with consistent gonioscopy and ultrasound biomicroscopy of the ciliary cleft, with no other ocular disease. Patients were divided into 3 groups, corresponding to their pre-operative IOP (group 1 ranging from 21 to 30 mm Hg, group 2 from 31 to 40 and group 3 for 40 and above). Ciliary process sonication was achieved with a probe containing one high-frequency transducer operating at 21 MHz during 5 seconds. Six sites were treated in patients from group 1, 8 in group 2, 10 in group 3, under general anesthesia. Post-operative treatment consisted of systemic meloxicam and topical carbonic anhydrase inhibitors, beta-blockers and prostaglandins analogues. No intraoperative complications were observed. Conjunctival hyperaemia occurred in eyes from group 2 (66%) and 3 (100%). Conjunctival burns were visible in 2 patients from group 3. One patient from group 3 experienced a hypertensive spike during the first hours post-op with associated pain. The hypotensive effect of HIFU was observed in all groups. Normotensive IOP (≤20 mm Hg) was reached in all patients until the last recheck at 6 months post op. Despite the small number of patients included in the study, HIFU appears to be a promising option for the management of PG in dogs.

Diseases of the retina and the optic nerve associated with obstructive sleep apnea.

Many associations between ocular disorders and obstructive sleep apnea (OSA) have been studied, such as nonarteritic anterior ischemic optic neuropathy, glaucoma, papilledema, retinal vein occlusion, eyelid hyperlaxity, lower-eyelid ectropion and recurrent corneal erosions. The objective of this review is to synthetize the possible vascular disorders of the retina and the optic nerve associated with sleep apnea patients and to discuss the underlying pathophysiological hypotheses. Main mechanisms involved in the ocular complications of OSA are related to intermittent hypoxia, sympathetic system activation, oxidant stress, and deleterious effects of endothelin 1. The main evidence-based medicine data suggest that OSA should be screened in patients with ischemic optic neuropathy and diabetic retinopathy. The effect of OSA treatment and emerging therapies are discussed.

Eye Size, Fovea, and Foraging Ecology in Accipitriform Raptors.

Birds with larger eyes are predicted to have higher spatial resolution because of their larger retinal image. Raptors are well known for their acute vision, mediated by their deep central fovea. Because foraging strategies may demand specific visual adaptations, eye size and fovea may differ between species with different foraging ecology. We tested whether predators (actively hunting mobile prey) and carrion eaters (eating dead prey) from the order Accipitriformes differ in eye size, foveal depth, and retinal thickness using spectral domain optical coherence tomography and comparative phylogenetic methods. We found that (1) all studied predators (except one) had a central and a temporal fovea, but all carrion eaters had only the central fovea; (2) eye size scaled with body mass both in predators and carrion eaters; (3) predators had larger eyes relative to body mass and a thicker retina at the edge of the fovea than carrion eaters, but there was no difference in the depth of the central fovea between the groups. Finally, we found that (4) larger eyes generally had a deeper central fovea. These results suggest that the visual system of raptors within the order Accipitriformes may be highly adapted to the foraging strategy, except for the foveal depth, which seems mostly dependent upon the eye size.

Compact Laser Doppler Flowmeter (LDF) Fundus Camera for the Assessment of Retinal Blood Perfusion in Small Animals.

PURPOSE: Noninvasive techniques for ocular blood perfusion assessment are of crucial importance for exploring microvascular alterations related to systemic and ocular diseases. However, few techniques adapted to rodents are available and most are invasive or not specifically focused on the optic nerve head (ONH), choroid or retinal circulation. Here we present the results obtained with a new rodent-adapted compact fundus camera based on laser Doppler flowmetry (LDF). METHODS: A confocal miniature flowmeter was fixed to a specially designed 3D rotating mechanical arm and adjusted on a rodent stereotaxic table in order to accurately point the laser beam at the retinal region of interest. The linearity of the LDF measurements was assessed using a rotating Teflon wheel and a flow of microspheres in a glass capillary. In vivo reproducibility was assessed in Wistar rats with repeated measurements (inter-session and inter-day) of retinal arteries and ONH blood velocity in six and ten rats, respectively. These parameters were also recorded during an acute intraocular pressure increase to 150 mmHg and after heart arrest (n = 5 rats). RESULTS: The perfusion measurements showed perfect linearity between LDF velocity and Teflon wheel or microsphere speed. Intraclass correlation coefficients for retinal arteries and ONH velocity (0.82 and 0.86, respectively) indicated strong inter-session repeatability and stability. Inter-day reproducibility was good (0.79 and 0.7, respectively). Upon ocular blood flow cessation, the retinal artery velocity signal substantially decreased, whereas the ONH signal did not significantly vary, suggesting that it could mostly be attributed to tissue light scattering. CONCLUSION: We have demonstrated that, while not adapted for ONH blood perfusion assessment, this device allows pertinent, stable and repeatable measurements of retinal blood perfusion in rats.

Use of bovine pericardium (Tutopatch®) graft for surgical repair of deep melting corneal ulcers in dogs and corneal sequestra in cats.

OBJECTIVE: To evaluate the efficacy of bovine pericardium (BP) graft in the treatment of deep melting corneal ulcers in three dogs and corneal sequestra in three cats. PROCEDURE: Three dogs with keratomalacia affecting the deep third of the stroma and three cats with corneal sequestrum were evaluated and underwent surgery. Following keratectomy, BP material was placed into the keratectomy bed and sutured to the recipient cornea with 9/0 polyglactin suture material. Postoperative treatment with topical and systemic antibiotics, systemic nonsteroidal anti-inflammatory agents, and topical atropine was prescribed. Follow-up examinations were carried out 1, 2 weeks, 1 and 2 months after the surgery and consisted of a complete ophthalmic examination. Optical coherence tomography (OCT) was performed 1 and 2 months after the surgery in one dog and in one cat. RESULTS: At 1 week, corneal neovascularization was present around the BP graft in all cases. Four weeks after the BP graft, in two dogs and in all cats, the vascularization was regressing and the graft was integrated into the cornea, which was regaining transparency. Topical treatment with anti-inflammatory agents was then prescribed for 2 weeks. Two months after the surgery, 5 of 6 corneas in two dogs and three cats had healed with focal corneal scarring. The remaining dog had progression of the keratomalacia involving the deep BP graft that required additional surgery, but became blind. CONCLUSION: Bovine pericardium graft offers a promising option for surgical reconstruction of the cornea following keratectomy for the management of corneal ulcers and sequestra.

Effect of mydriasis induced by topical instillations of 0.5% tropicamide on the anterior segment in normotensive dogs using ultrasound biomicroscopy.

OBJECTIVE: To determine the effect of 0.5% tropicamide and the resultant mydriasis on the anterior segment in normotensive dogs using ultrasound biomicroscopy. PROCEDURE: Twenty-four dogs without ocular disease underwent ultrasound biomicroscopic examination of both the eyes under general anesthesia. Pharmacologic mydriasis was induced in the right eye of each dog by the topical instillation of 0.5% tropicamide. Ultrasound biomicroscopic examinations were performed and the anatomical parameters of the anterior segment were evaluated including the geometric iridocorneal angle (ICA) - formed by the plane of the iris root and the internal corneoscleral limbus - the width of the opening of the ciliary cleft (CC), the width of the mid-CC, the length of the CC, and the anterior chamber (AC) depth (distance between the corneal endothelium and the anterior lens capsule). RESULTS: Mydriasis resulting from the topical use of 0.5% tropicamide is associated with an increase in the geometric ICA and a decrease in the opening of the CC, without any effect on the width of the mid-CC, or on the length of the CC, or on the AC depth. CONCLUSIONS: Mydriasis induced by topical 0.5% tropicamide results in modification of the anterior segment which may influence the drainage of aqueous humor.