Cell therapy in the cornea: The emerging role of microenvironment.

The cornea is an ideal testing field for cell therapies. Its highly ordered structure, where specific cell populations are sequestered in different layers, together with its accessibility, has allowed the development of the first stem cell-based therapy approved by the European Medicine Agency. Today, different techniques have been proposed for autologous and allogeneic limbal and non-limbal cell transplantation. Cell replacement has also been attempted in cases of endothelial cell decompensation as it occurs in Fuchs dystrophy: injection of cultivated allogeneic endothelial cells is now in advanced phases of clinical development. Recently, stromal substitutes have been developed with excellent integration capability and transparency. Finally, cell-derived products, such as exosomes obtained from different sources, have been investigated for the treatment of severe corneal diseases with encouraging results. Optimization of the success rate of cell therapies obviously requires high-quality cultured cells/products, but the role of the surrounding microenvironment is equally important to allow engraftment of transplanted cells, to preserve their functions and, ultimately, lead to restoration of tissue integrity and transparency of the cornea.

Aprepitant Restores Corneal Sensitivity and Reduces Pain in DED.

Purpose: This study aims to assess the efficacy of two aprepitant formulations (X1 and X2), in a preclinical model of dry eye disease (DED) induced by benzalkonium chloride (BAK). Methods: Two aprepitant formulations were tested on 7 to 8-week-old male mice for their efficacy. In vivo corneal fluorescein staining assessed epithelial damage as the primary end point on days 0, 3, 5, 7, 9, 12, and 14 using slit-lamp microscopy. The DED model was induced with 0.2% BAK twice daily for the first week and once daily for the next week. Mice were randomly assigned to 5 treatment groups: Aprepitant X1 (n = 10) and X2 (n = 10) formulation, 2 mg/mL dexamethasone (n = 10), control vehicle X (n = 10), 0.2% hyaluronic acid (n = 10), or no treatment (n = 10). Eye wiping, phenol red, and Cochet Bonnet tests assessed ocular pain, tear fluid secretion, and nerve function. After 7 days, the mice were euthanized to quantify leukocyte infiltration and corneal nerve density. Results: Topical aprepitant X1 reduced BAK-induced corneal damage and pain compared to gel vehicle X (P = 0.007) and dexamethasone (P = 0.021). Aprepitant X1 and X2 improved corneal sensitivity versus gel vehicle X and dexamethasone (P < 0.001). Aprepitant X1 reduced leukocyte infiltration (P < 0.05) and enhanced corneal nerve density (P < 0.001). Tear fluid secretion remained statistically unchanged in both the X1 and X2 groups. Conclusions: Aprepitant formulation X1 reduced pain, improved corneal sensitivity and nerve density, ameliorated epitheliopathy, and reduced leukocyte infiltration in male mouse corneas. Translational Relevance: Aprepitant emerges as a safe, promising therapeutic prospect for the amelioration of DED's associated symptoms.

Anterior segment involvement in vitreoretinal lymphoma: clinical manifestations, molecular findings and in vivo confocal microscopy.

BACKGROUND: Intermediate and posterior manifestations of vitreoretinal lymphoma (VRL) are well characterised. However, there is limited information on anterior segment involvement in VRL. This study aimed to describe the anterior manifestations of VRL, and their association with molecular testing. METHODS: Retrospective analysis of patients with biopsy-proven VRL. Study variables included anterior segment manifestations, findings from slit-lamp photos and in vivo confocal microscopy (IVCM) when available. MYD88 L265P mutation and cytology in the aqueous humour, retinal and systemic findings were also analysed. RESULTS: The analysis included 108 eyes of 55 VRL patients. Anterior segment involvement was present in at least one visit in 55 eyes (51%) of 33 patients (60%); it included keratic precipitates (dendritiform with branching and irregular margins in 33 eyes, dust-like in 16 eyes and large granulomatous in 12 eyes), cells in the anterior chamber (51 eyes) and posterior synechiae (2 eyes). IVCM was available for 41 eyes and showed different morphologies of keratic precipitates, including floral, spikes and mulberry patterns (66%, 56% and 20%, respectively). MYD88 L265P mutation in the aqueous humour was detected in 10/21 (48%) eyes with no anterior segment involvement and 24/37 (65%) eyes with anterior segment involvement. CONCLUSIONS: Anterior segment manifestations are often present in VRL and include dendritiform and dust-like keratic precipitates. IVCM in VRL can identify different patterns associated with keratic precipitates. MYD88 L265P mutation in the aqueous humour of VRL patients can also be found in eyes without significant anterior segment involvement.

Topical fosaprepitant for the treatment of ocular pain and inflammation.

Purpose: To assess whether topical administration of fosaprepitant improves intractable chronic ocular pain and inflammation. Methods: We report three clinical cases of female patients with drug-resistant ocular pain associated with inflammatory diseases of the ocular surface. The patients were treated for 3 (case 1) and 4 (cases 2-3) weeks with fosaprepitant eyedrops (0.1 mg/mL for case 1; 10 mg/mL for case 2-3). Patients were then followed up for at least 3 weeks. We measured ocular pain with the Visual Analogue Scale (VAS), the Ocular Surface Disease Index (OSDI), and corneal sensitivity with the Cochet-Bonnet esthesiometry. Slit-lamp photography and corneal confocal imaging were used to assess ocular surface integrity/conjunctival hyperemia and corneal nerve morphology, respectively. Results: All three patients had severe ocular pain (score higher than 6/10 VAS scale). All patients reported a significant improvement in ocular pain after 1 week of treatment. We also observed reduced corneal epitheliopathy (case 1) and conjunctival hyperemia (cases 1-2). In two patients (cases 2-3) the treatment was repeated after 1 year and 9 weeks, respectively, and pain reduction was similar in magnitude to what we observed after the first administration. Conclusions: Topical administration of fosaprepitant ameliorates ocular pain and clinical symptoms in three patients with intractable ocular pain associated with inflammatory diseases of the ocular surface, without adverse effects. Importance: Fosaprepitant instillation holds promise as a treatment of chronic ocular pain, an area of unmet medical need.

Managing the ocular surface after glaucoma filtration surgery: an orphan topic.

BACKGROUND: Ocular surface (OS) disorders before glaucoma filtration surgery (GFS) have been considered to play a crucial role influencing the surgical outcome. Conversely, the impact of surgery itself on the OS is almost completely overlooked, though evidence suggest that ocular surface disease (OSD) may be induced in patients by GFS. This review analyzes the determinants involved in the OSD development after GFS, the clinical features and related consequences, the main diagnostic hallmarks, as well as the therapeutic strategies for its management. METHODS: The PubMed database was utilized for the literature examination. Keywords that were searched included ocular surface disease, glaucoma filtration surgery, filtration bleb, post-surgical management, and quality of life. RESULTS: After GFS, OSD is promoted by peri- and post-operative factors, such as the filtration bleb (FB) development, combined surgical approach with phacoemulsification, the use of antifibrotic agents and the reintroduction of antiglaucoma medications. This particular form of OSD that present similar clinical features to mild to moderate dry eye, can be named as post-glaucoma surgery-OSD (PGS-OSD). PGS-OSD may negatively affect the FB functionality, thus potentially hindering the disease control, and significantly worsen the patient quality of life (QOL). CONCLUSIONS: Clinicians are encouraged to routinely include the OS evaluation after GFS and to consider proper management when the occurrence of PGS-OSD worsen the patient's QOL or exert negative effects to the FB functionality. An outline summarizing the main risk factors and the most appropriate therapeutic options to mitigate the PGS-OSD was proposed to support the routine practice.

Beauveria bassiana Keratitis: A Case Series and Review of Literature.

BACKGROUND: Beauveria bassiana is a filamentous fungus commonly used as an insecticide that rarely causes keratitis. METHODS: Patients affected by Beauveria bassiana keratitis were retrospectively recruited at San Raffaele Hospital (Milan, Italy) between 2020 and 2022. All subjects underwent comprehensive ophthalmic evaluation, including in vivo confocal microscopy (IVCM) and microbiologic examination of corneal scrapings. Beauveria bassiana was identified using 18S rDNA targeted PCR. RESULTS: Four eyes of four patients (51 ± 8.8 years old) were evaluated. The main risk factors were soft contact lens wear (75%) and trauma with vegetative matter (50%). A superficial infiltrate was displayed in the majority of patients. Three cases (75%) showed hyphae on IVCM. All patients showed clinical improvement after topical antifungal therapy, although mostly through a combination of two antifungals (75%). One patient with a deeper infection required a systemic antifungal agent after one month of topical therapy. All cases required debridement to reduce the microbial load and enhance drug penetration. All patients experienced keratitis resolution following medical treatment (average: 3.3 months). CONCLUSIONS: The identification of risk factors and the early diagnosis of Beauveria bassiana keratitis are fundamental in order to avoid its penetration in the deeper corneal stromal layers. Topical antifungal drugs, possibly accompanied by ulcer debridement, may be a successful treatment if instilled from the early phases of the disease.

Topical formulations of Aprepitant are safe and effective in relieving pain and inflammation, and drive neural regeneration.

PURPOSE: To test long-term ocular toxicity and analgesic/anti-inflammatory efficacy of two novel ocular formulations of neurokinin 1 receptor (NK1R) antagonist Aprepitant. METHODS: for toxicity studies, two Aprepitant formulations (X and Y) were tested on C57BL/6 N mice. Gold standards were 0.4% Oxybuprocaine, 0.1% Diclofenac, or saline. For efficacy studies, C57BL/6 N mice underwent corneal alkali burn, and then received Aprepitant formulation X, Dexamethasone or saline. Eye-drops were applied 3 times/day for 90 days (toxicity) and 14 days (efficacy). Stromal opacity, corneal epithelial damage, nociception and sensitivity were assessed in vivo. The eye-wiping test and corneal sensitivity were assessed to evaluate analgesic efficacy and nerve function. At the end of the experiments mice were euthanized, and corneas were dissected for immunohistochemistry and RT-PCR analyses. RESULTS: In normal mice, formulation X was not toxic when topically administered for 90 days. Formulation Y was associated with increased leukocyte infiltration in the cornea (p < 0.001). X1 and X2 formulations significantly reduced corneal pain, as Diclofenac and Oxybuprocaine, but did not reduce corneal sensitivity. Formulation Y, instead, was not analgesic at any time point. In the alkali burn model, X1 and X2 formulation enhanced epithelial damage recovery, and reduced inflammation both at day 7 and 14. Moreover, formulation X showed a stronger analgesic effect when compared to the saline and Dexamethasone groups (p < 0.01). Finally, formulation X1 and X2 restored corneal sensitivity by promoting corneal nerve regeneration. CONCLUSIONS: Aprepitant X formulation is a promising candidate for the treatment of pain associated with inflammation of the ocular surface.

Safety and Efficacy of Corneal Cross-Linking in Patients Affected by Keratoconus: Long-Term Results.

The present study evaluated the effectiveness and safety of corneal collagen cross-linking (CXL). A total of 886 eyes with progressive keratoconus were enrolled in a retrospective cohort study in a tertiary care university hospital. CXL was performed using a standard epithelium-off Dresden protocol. Visual outcomes, maximum keratometry (Kmax), demarcation line measurements, and complications were recorded. Visual outcomes and keratometric data were analyzed in a subgroup comprising 610 eyes. Uncorrected distance visual acuity (UDVA) improved from 0.49 ± 0.38 LogMAR to 0.47 ± 0.39 LogMAR (p = 0.03, n = 610) three years after the procedure, while corrected distance visual acuity (CDVA) improved from 0.15 ± 0.14 LogMAR to 0.14 ± 0.15 LogMAR (p = 0.007, n = 610). A significant reduction of Kmax from 56.28 ± 6.10 to 54.98 ± 6.19 (p < 0.001, n = 610) was observed three years after CXL. In five eyes (0.82%, 5/610) keratoconus progression continued after CXL. Three eyes were retreated successfully with documented refractive and topographic stability after five years. In the 35 eyes that completed 10 years of follow-up, mean visual acuity and topographic parameters remained stable. In conclusion, CXL is a safe and effective treatment for avoiding keratoconus progression. Long-term data are encouraging, supporting a high safety profile for this procedure.

Neurosensory abnormalities and stability of a mouse model of dry eye disease.

This study aimed to use a mouse model of dry eye disease (DED) induced by topical administration of benzalkonium chloride (BAK) and assess its stability and the presence of neurosensory abnormalities, including ocular pain. Eight-week-old C57BL6/6 N male mice were used in this study. Mice were treated with 10 µL of 0.2% BAK dissolved in artificial tears (AT), administered twice daily for 7 days. After one week, animals were randomized into two groups: one was administered with 0.2% BAK in AT once per day for 7 days, while the other was not further treated. Corneal epitheliopathy was quantified at days 0, 3, 7, 12, and 14. Moreover, tear secretions, corneal nociception, and corneal nerve integrity were measured after BAK treatment. After sacrifice, corneas were dissected to assess nerve density and leukocyte infiltration by immunofluorescence. Topical BAK instillation for 14 days significantly increased corneal fluorescein staining (p < 0.0001) compared to day 0. On the other hand, interruption of BAK instillation was associated with improvement of corneal epitheliopathy (day 12, p < 0.0001; day 14, p < 0.001). BAK treatment increased ocular pain (p < 0.0001) and resulted in a significant increase in leukocyte infiltration in the cornea (p < 0.01). Moreover, corneal sensitivity was reduced (p < 0.0001), together with corneal nerve density (p < 0.0001) and tear secretion (p < 0.0001). One week twice a day, followed by one additional week once a day, of 0.2% BAK topical administration induces stable clinical and histological signs of DED, which is associated with neurosensory abnormalities, including pain.

Future directions in managing aniridia-associated keratopathy.

Congenital aniridia is a panocular disorder that is typically characterized by iris hypoplasia and aniridia-associated keratopathy (AAK). AAK results in the progressive loss of corneal transparency and thereby loss of vision. Currently, there is no approved therapy to delay or prevent its progression, and clinical management is challenging because of phenotypic variability and high risk of complications after interventions; however, new insights into the molecular pathogenesis of AAK may help improve its management. Here, we review the current understanding about the pathogenesis and management of AAK. We highlight the biological mechanisms involved in AAK development with the aim to develop future treatment options, including surgical, pharmacological, cell therapies, and gene therapies.

TFOS Lifestyle: Impact of lifestyle challenges on the ocular surface.

Many factors in the domains of mental, physical, and social health have been associated with various ocular surface diseases, with most of the focus centered on aspects of dry eye disease (DED). Regarding mental health factors, several cross-sectional studies have noted associations between depression and anxiety, and medications used to treat these disorders, and DED symptoms. Sleep disorders (both involving quality and quantity of sleep) have also been associated with DED symptoms. Under the domain of physical health, several factors have been linked to meibomian gland abnormalities, including obesity and face mask wear. Cross-sectional studies have also linked chronic pain conditions, specifically migraine, chronic pain syndrome and fibromyalgia, to DED, principally focusing on DED symptoms. A systematic review and meta-analysis reviewed available data and concluded that various chronic pain conditions increased the risk of DED (variably defined), with odds ratios ranging from 1.60 to 2.16. However, heterogeneity was noted, highlighting the need for additional studies examining the impact of chronic pain on DED signs and subtype (evaporative versus aqueous deficient). With respect to societal factors, tobacco use has been most closely linked to tear instability, cocaine to decreased corneal sensitivity, and alcohol to tear film disturbances and DED symptoms.

Evaluation of a Post-Operative Therapy Protocol after Epithelium-Off Corneal Cross-Linking in Patients Affected by Keratoconus.

A large retrospective study evaluated the safety of a post-operative therapy protocol after epithelium-off corneal collagen cross-linking (CXL). In total, 1703 eyes of the 1190 patients with progressive keratoconus were enrolled in a retrospective cohort study in a tertiary care university hospital. CXL was performed using a standardized technique (Dresden protocol: 0.1% riboflavin solution containing dextran 20% for 30 min during the soaking phase followed by 30-min ultraviolet A irradiation (3 mW/cm2)). Postoperatively, a bandage contact lens was applied, and therapy included a topical fluoroquinolone antibiotic until the epithelium healed, followed by topical fluorometholone treatment for three weeks. Post-operative complications were recorded and analyzed. No cases of infectious keratitis occurred, whereas peripheral sterile infiltrates were observed in 1.17% of cases. Trace haze was typically present but did not have an impact on visual acuity. In fifteen cases (0.88%), visually significant anterior stromal opacity developed. Mild signs of dry eye were observed in 22 eyes (1.29%). The present study demonstrates that a post-operative treatment protocol including fluoroquinolone antibiotics and a BCL in the first phase until complete epithelial healing, followed by a three-week period of topical steroid treatment is safe and not associated with the development of microbial keratitis.

Vitamin D Supplementation Impacts Systemic Biomarkers of Collagen Degradation and Copper Metabolism in Patients With Keratoconus.

Purpose: To evaluate the impact of vitamin D (Vit D) supplementation on systemic biomarkers of collagen degradation, inflammation, oxidative stress, and copper metabolism in adolescent patients with keratoconus (KC). Methods: This was a prospective observational pilot study. Twenty patients (age range, 16-19 years) presenting KC and Vit D insufficiency (<30 ng/mL) were included. Vit D supplementation was prescribed by their general practitioner as per the standard of care. Patients were followed up for 12 months. At each visit, best spectacle-corrected visual acuity (BSCVA), maximal keratometry (Kmax), and thinnest corneal thickness (TCT) were evaluated. The primary outcome of the study was the proportion of patients with Kmax progression of less than 1 D throughout the 12-month follow-up time. Blood samples were collected at different time points to evaluate Vit D levels and systemic markers of collagen degradation, inflammation, oxidative stress, and copper metabolism by ELISA or RT-PCR. Results: Lower Vit D levels in the plasma were correlated with higher levels of systemic biomarkers of collagen degradation. Vit D supplementation increased the cell availability of copper. Moreover, stabilization of KC progression was found in 60% of patients (72% of eyes) after 12 months with Vit D supplementation. BSCVA, Kmax, and TCT rates remained stable during the observation period. Conclusions: Our findings support that Vit D administration could affect ocular and systemic biomarkers in KC and illuminate a possible mechanism that can be used to develop new treatment alternatives. Translational Relevance: Although KC therapy currently relies exclusively on surgical procedures, Vit D supplementation may offer a non-invasive and inexpensive alternative with minimal associated side effects.

Modulating the tachykinin: Role of substance P and neurokinin receptor expression in ocular surface disorders.

Substance P (SP) is a tachykinin expressed by various cells in the nervous and immune systems. SP is predominantly released by neurons and exerts its biological and immunological effects through the neurokinin receptors, primarily the neurokinin-1 receptor (NK1R). SP is essential for maintaining ocular surface homeostasis, and its reduced levels in disorders like diabetic neuropathy disrupt the corneal tissue. It also plays an essential role in promoting corneal wound healing by promoting the migration of keratocytes. In this review, we briefly discuss the structure, expression, and function of SP and its principal receptor NK1R. In addition, SP induces pro-inflammatory effects through autocrine or paracrine action on the immune cells in various ocular surface pathologies, including dry eye disease, herpes simplex virus keratitis, and Pseudomonas keratitis. We provide an in-depth review of the pathogenic role of SP in various ocular surface diseases and several new approaches developed to counter the immune-mediated effects of SP either through modulating its production or blocking its target receptor.

Corneal endothelial cell reduction and increased Neurokinin-1 receptor expression in a graft-versus-host disease preclinical model.

The aim of this work was to assess corneal endothelial morphology in a well-established acute graft-versus-host disease (GVHD) murine model and to quantify the expression of neurokinin-1 receptor (NK1R) in the corneal endothelium during ocular GVHD (oGVHD). Pre-conditioning was performed in BALB/c using myeloablative total body irradiation. Subsequently, allogeneic bone marrow transplantation was performed without (BM) or with mature T cells (BM + T). Corneal transparency was monitored with in vivo biomicroscopy. After sacrifice, corneal thickness and endothelial cell number were measured, and the expression of NK1R was investigated in the corneal endothelium through immunofluorescence and quantified by immunohistochemistry. Mice presenting oGVHD showed a significant reduction in endothelial cell number compared to control animals (p < 0.0001). NK1R expression was significantly increased in oGVHD mice endothelium (p < 0.05). Corneal transparency and thickness were unchanged in all groups. Our results suggest that oGVHD affects the corneal endothelium, inducing a reduction of the cell number, and that this is associated with increased expression of the pro-inflammatory marker NK1R.

Corneal and Epidermal Nerve Quantification in Chemotherapy Induced Peripheral Neuropathy.

Chemotherapy-induced neurotoxicity is an increasingly recognized clinical issue in oncology. in vivo confocal microscopy (IVCM) of corneal nerves has been successfully used to diagnose peripheral neuropathies, including diabetic neuropathy. The purpose of this study was to test if the combination of corneal nerve density and morphology assessed by IVCM is useful to monitor the neurotoxic effects of chemotherapy compared to epidermal nerve quantification. Overall, 95 adult patients with different cancer types were recruited from the oncology and hematology departments of the San Raffaele Hospital. Neurological examination, including clinical Total Neuropathy Score, and in vivo corneal confocal microscopy (IVCM), were performed before and after chemotherapy. In a group of 14 patients, skin biopsy was performed at the first and last visit. In the group of 14 patients who underwent both skin biopsy and corneal nerve imaging, clinical worsening (+69%, p = 0.0018) was paralleled by corneal nerve fiber (CNF) density reduction (-22%, p = 0.0457). Clinical Total neuropathy score significantly worsened from the first to the last visit (+62%, p < 0.0001). CNF length was not significantly reduced overall. However, CNF density/tortuosity ratio significantly decreased after therapy. Correlation analysis showed that the CNF density/tortuosity ratio was also correlated with the number of chemotherapy cycles (r = -0.04790, P = 0.0009). Our data confirm that in vivo corneal confocal microscopy is a helpful, non-invasive tool which shows promise for the diagnosis of chemotherapy-induced peripheral neuropathies. IVCM could allow a rapid, reproducible and non-invasive quantification of peripheral nerve pathology in chemotherapy-associated neuropathy.

Substance P/neurokinin-1 receptor pathway blockade ameliorates limbal stem cell deficiency by modulating mTOR pathway and preventing cell senescence.

Severe ocular surface diseases can lead to limbal stem cell deficiency (LSCD), which is accompanied by defective healing. We aimed to evaluate the role of the substance P (SP)/neurokinin-1 receptor (NK1R) pathway in corneal epithelium wound healing in a pre-clinical model of LSCD. SP ablation or NK1R blockade significantly increased epithelial wound healing (p < 0.001) and corneal transparency (p < 0.001), compared with wild type (WT). In addition, a reduced number of infiltrating goblet and conjunctival cells (p < 0.05) and increased number of epithelial stem cells (p < 0.01), which also expressed NK1R, was observed. The mammalian target of rapamycin (mTOR) pathway was significantly inhibited (p < 0.05) and expression of γH2AX was significantly reduced (p < 0.05) after SP ablation. These results suggest that excessive expression of SP is associated with LSCD and results in accelerated senescence and exhaustion of residual stem cells. Topical treatment with NK1R antagonist ameliorates clinical signs associated with LSCD and could be used as an adjuvant treatment in LSCD.