Combinative approach of transzonular triamcinolone-moxifloxacin and perioperative drops to minimize postoperative complications of cataract surgery.

AIM: To investigate the effectiveness of combination therapy with transzonular triamcinolone-moxifloxacin and conventional perioperative drops in reducing postoperative complications of cataract surgery. METHODS: Electronic medical records of cataract surgery patients (single surgeon) were reviewed from January 2018 to September 2021. The rate of postoperative complications including prolonged and/or recurrent postoperative inflammation, endophthalmitis, cystoid macular edema (CME), and intraocular pressure (IOP) was compared between the patients receiving combinative therapy and patients receiving drops only. RESULTS: Totally 596 patients and 1057 eyes (Combinative-Therapy group 493 and Drop-Only group 564) were included in this study. Using combination therapy reduced the relative risk of postoperative inflammation by 26.9% (16.6% Combinative-Therapy vs 22.7% Drop-Only, P=0.013). The incidence of endophthalmitis was 0 in Combinative-Therapy group vs 0.5% in Drop-Only group (relative risk reduction 100%), although not statistically significant (P=0.10). The incidence of severe IOP spikes was not significantly different between Combinative-Therapy (2.4%) and Drop-Only (1.6%) groups (P=0.33). The relative risk of postoperative CME was 51.4% less in three months follow up visit in Combinative-Therapy group, although not statistically significant (P=0.07). The visual outcome 1-month postop. (best corrected visual acuity) was significantly better in Combinative-Therapy (logMAR 0.10) compared to Drop-Only (logMAR 0.14) groups (P=0.02) while the baseline visual acuity was not significantly different. CONCLUSION: The combinative approach of transzonular triamcinolone-moxifloxacin plus perioperative eyedrops is an effective method to minimize postoperative inflammation, with better visual outcomes. It could potentially reduce the risk of postoperative endophthalmitis and CME (near-significant P-values; larger studies could analyze better considering low incidence).

A population-based prospective study on obesity-related non-communicable diseases in northern Iran: rationale, study design, and baseline analysis.

Background: Iran is facing an epidemiological transition with the increasing burden of non-communicable diseases, such as obesity-related disorders and cardiovascular diseases (CVDs). We conducted a population-based prospective study to assess the prevalence and incidence rates of CVDs and obesity-related metabolic disorders and to evaluate the predictive ability of various CVD risk assessment tools in an Iranian population. Method: We enrolled 5,799 participants in Amol, a city in northern Iran, in 2009-2010 and carried out the first repeated measurement (RM) after seven years (2016-2017). For all participants, demographic, anthropometric, laboratory, hepatobiliary imaging, and electrocardiography data have been collected in the enrollment and the RM. After enrollment, all participants have been and will be followed up annually for 20 years, both actively and passively. Results: We adopted a multidisciplinary approach to overcome barriers to participation and achieved a 7-year follow-up success rate of 93.0% with an active follow-up of 5,394 participants aged 18-90 years. In the RM, about 64.0% of men and 81.2% of women were obese or overweight. In 2017, about 16.2% and 5.2% of men had moderate or severe non-alcoholic fatty liver disease, while women had a significantly higher prevalence of metabolic syndrome (35.9%), and type 2 diabetes mellitus (20.9%) than men. Of 160 deceased participants, 69 cases (43.1%) died due to CVDs over seven years. Conclusion: The most prevalent obesity-related chronic disease in the study was metabolic syndrome. Across the enrollment and RM phases, women exhibited a higher prevalence of obesity-related metabolic disorders. Focusing on obesity-related metabolic disorders in a population not represented previously and a multidisciplinary approach for enrolling and following up were the strengths of this study. The study outcomes offer an evidence base for future research and inform policies regarding non-communicable diseases in northern Iran.

Prognostic factors of the synovial sarcoma of the extremities: imaging does matter.

OBJECTIVES: Synovial sarcomas (SS) of the extremities are rare soft tissue sarcomas that are more common in young adults. We deciphered the imaging phenotype of SS with the aim to determine if imaging could provide an incremental value to currently known prognostic factors (PF)-age and histological grade-to predict long-term overall survival (OS). METHODS: This retrospective multicenter study included consecutive pediatric and adult patients with synovial sarcomas of the extremities from December 2002 to August 2020. Inclusion criteria were (i) a follow-up greater than 5 years and (ii) available pre-therapeutic MRI. A subset analysis included MRI and CT-scan. Clinical, pathological, and imaging variables were collected in all patients. The primary endpoint was to evaluate the association of these variables with OS using univariate and multivariate Cox regressions. RESULTS: Out of 428 patients screened for eligibility, 98 patients (mean age: 37.1 ± 15.2 years) were included (MRI: n = 98/98, CT scan: n = 34/98; 35%). The median OS was 75.25 months (IQR = 55.50-109.12) and thirty-six patients (n = 36/98;37%) died during follow-up. The recurrence rate was 12.2% (n =12/98). SS lesions were mostly grade 2 (57/98; 58%). On MRI, SS had a mean long-axis diameter of 67.5 ± 38.3 mm. On CT scan, 44% (15/34) were calcified. Grade (hazard ratio [HR] = 2.71; 95%CI = 1.30-5.66; p = 0.008), size of the lesions evaluated on MRI (HR = 1.02; 95% CI = 1.01-1.03; p < 0.001), and calcifications on CT scan (HR = 0.10; 95% CI = 0.02-0.50; p = 0.005) were independent PF of OS. CONCLUSIONS: This study demonstrated that imaging biomarkers can be used to predict long-term outcome in patients with SS. Strikingly, the presence of calcifications on CT scan is associated with favorable outcome and provides an incremental value over existing PF such as age, grade, and size. KEY POINTS: • Beyond its diagnostic value, MRI is a pre-operative prognostic tool in synovial sarcomas of the extremities since the size of the lesion is an important prognostic factor. • Calcifications on CT scans are independently and significantly associated with prolonged overall survival.

Papillary conjunctivitis presenting months after permanent eyebrow tattooing.

Purpose: To present a case of tattoo side effects not limited to the tattoo site and rise an alarm regarding using non-FDA-approved products. Observations: A 30-year-old female presented with bilateral ocular pain, dryness, and itching. The ocular exam showed bilateral injection and edema of the superior palpebral and bulbar conjunctiva. Several 1-2 mm dark pigmented lesions and papillae coursing along the upper palpebral conjunctival lid margin and 5 mm above the margin were found in both eyes. The ocular surface was dry with diffuse superficial punctate keratitis. The biopsy report showed granular foreign material in the dermis. SOX-10 and MART-1 immunostaining highlighted melanocyte distribution and the sample was diagnosed as exogenous pigment consistent with tattoo ink by the pathologist. On further investigation following the pathology report, the patient stated that she got bilateral permanent eyebrow tattoos 4 months before presentation in a country other than the United States, and she was not aware about the standards of the ink used, nor the certification of the person performing the tattoo. The patient denied any type of tattoo or manipulation on the eyes or orbit, including sclera or conjunctivae. Conclusions: Importance: The complications of periorbital tattooing are not limited to the point tattoo location and can potentially spread to the nearby segments. It is notable that there is no FDA approved tattoo ink available, even with a certified tattoo artist performing the tattoo, the risks of inflammation, infection, and other side effects are still present.

Long-term retinal protection by MEK inhibition in Pax6 haploinsufficiency mice.

Aniridia is a panocular condition characterized by impaired eye development and vision, which is mainly due to the haploinsufficiency of the paired-box-6 (PAX6) gene. Like what is seen in aniridia patients, Pax6-deficient mice Pax6Sey-Neu/+ exhibit a varied degree of ocular damage and impaired vision. Our previous studies showed that these phenotypes were partially rescued by PD0325901, a mitogen-activated protein kinase kinase (MEK or MAP2K) inhibitor. In this study, we assessed the long-term efficacy of PD0325901 treatment in retinal health and visual behavior. At about one year after the postnatal treatment with PD0325901, Pax6Sey-Neu/+ mice showed robust improvements in retina size and visual acuity, and the elevated intraocular pressure (IOP) was also alleviated, compared to age-matched mice treated with vehicles only. Moreover, the Pax6Sey-Neu/+ eyes showed disorganized retinal ganglion cell (RGC) axon bundles and retinal layers, which we termed as hotspots. We found that the PD treatment reduced the number and size of hotspots in the Pax6Sey-Neu/+ retinas. Taken together, our results suggest that PD0325901 may serve as an efficacious intervention in protecting retina and visual function in aniridia-afflicted subjects.

FDG-PET/CT of COVID-19 and Other Lung Infections.

While not conventionally used as the first-line modality, [18F]-2-fluoro-2-deoxy-D-glucose (FDG) - positron emission tomography/computed tomography (PET/CT) can identify infection and inflammation both earlier and with higher sensitivity than anatomic imaging modalities [including chest X-ray (CXR), computed tomography (CT), and magnetic resonance imaging (MRI)]. The extent of inflammation and, conversely, recovery within the lungs, can be roughly quantified on FDG-PET/CT using maximum standardized uptake value (SUVmax) values. The Coronavirus disease 2019 (COVID-19) pandemic has highlighted the value of FDG-PET/CT in diagnosis, elucidation of acute pulmonary and extrapulmonary manifestations, and long-term follow up. Similarly, many other pulmonary infections such as previously documented coronaviruses, aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mucormycosis, and typical/atypical mycobacterial infections have all been identified and characterized using FDG-PET/CT imaging. The goal of this review is to summarize the actual and potential benefits of FDG-PET/CT in the imaging of COVID-19 and other lung infections. Further research is necessary to determine the best indications and clinical applications of FDG-PET/CT, improve its specificity, and ultimately ascertain how this modality can best be utilized in the diagnostic work up of infectious pathologies.

The role of imaging techniques in understanding and evaluating the long-term pulmonary effects of COVID-19.

INTRODUCTION: Limited data exist regarding the long-term pulmonary sequelae of COVID-19. Identifying features utilizing multiple imaging modalities engenders a clearer picture of the illness's long-term consequences. AREAS COVERED: This review encompasses the common pulmonary findings associated with different imaging modalities during acute and late remission stages of COVID-19 pneumonia. EXPERT OPINION: Chest x-ray, a common preliminary diagnostic imaging technique, is not optimal for extended care due to limited tissue contrast resolution providing suboptimal assessment of pulmonary pathology and subtle interval changes. Ultrasound may be utilized on a case-by-case basis in certain patient populations, or in countries with limited resources. Chest CT's accessibility, high tissue contrast and spatial resolution make it the foremost modality for long-term COVID-19 follow-up. While MRI can viably monitor extrapulmonary disease due to its lack of radiation and high inherent soft-tissue contrast, it has limited pulmonary utility due to motion artifact and alveolar gas decreasing lung signal. Although 18F-FDG-PET/CT is costly and has limited specificity, it can provide molecular level data and inflammation quantification. Lung perfusion scintigraphy may also explain COVID-19 induced thromboembolic events and persistent dyspnea despite normal structural imaging and testing results. Correlating the long-term pulmonary findings of COVID-19 with each imaging modality is essential in elucidating the post-recovery course.

Preclinical Evaluation of the Safety and Efficacy of Cryopreserved Bone Marrow Mesenchymal Stromal Cells for Corneal Repair.

Purpose: Mesenchymal stromal cells (MSCs) have been shown to enhance tissue repair as a cell-based therapy. In preparation for a phase I clinical study, we evaluated the safety, dosing, and efficacy of bone marrow-derived MSCs after subconjunctival injection in preclinical animal models of mice, rats, and rabbits. Methods: Human bone marrow-derived MSCs were expanded to passage 4 and cryopreserved. Viability of MSCs after thawing and injection through small-gauge needles was evaluated by vital dye staining. The in vivo safety of human and rabbit MSCs was studied by subconjunctivally injecting MSCs in rabbits with follow-up to 90 days. The potency of MSCs on accelerating wound healing was evaluated in vitro using a scratch assay and in vivo using 2-mm corneal epithelial debridement wounds in mice. Human MSCs were tracked after subconjunctival injection in rat and rabbit eyes. Results: The viability of MSCs after thawing and immediate injection through 27- and 30-gauge needles was 93.1% ± 2.1% and 94.9% ± 1.3%, respectively. Rabbit eyes demonstrated mild self-limiting conjunctival inflammation at the site of injection with human but not rabbit MSCs. In scratch assay, the mean wound healing area was 93.5% ± 12.1% in epithelial cells co-cultured with MSCs compared with 40.8% ± 23.1% in controls. At 24 hours after wounding, all MSC-injected murine eyes had 100% corneal wound closure compared with 79.9% ± 5.5% in controls. Human MSCs were detectable in the subconjunctival area and peripheral cornea at 14 days after injection. Conclusions: Subconjunctival administration of MSCs is safe and effective in promoting corneal epithelial wound healing in animal models. Translational Relevance: These results provide preclinical data to support a phase I clinical study.

Predicting the aggressiveness of peripheral zone prostate cancer using a fractional order calculus diffusion model.

PURPOSE: To evaluate the performance of parameters D, ß, µ from the Fractional Order Calculus (FROC) model at differentiating peripheral zone (PZ) prostate cancer (PCa) MATERIAL AND METHODS: 75 patients who underwent targeted MRI-guided TRUS prostate biopsy within 6 months of MRI were reviewed retrospectively. Regions of interest (ROI) were placed on suspicious lesions on MRI scans. ROIs were then correlated to pathological results based on core biopsy location. The final tumor count is a total: 23 of GS 6 (3 + 3), 36 of GS 7 (3 + 4), 18 of GS 7 (4 + 3), and 19 of GS ≥ 8. Diffusion-weighted imaging (DWI) scans were fitted into the FROC and monoexponential model to calculate ADC and FROC parameters: anomalous diffusion coefficient D, intravoxel diffusion heterogeneity ß, and spatial parameter µ. The performance of FROC parameters and ADC at differentiating PCa grade was evaluated with receiver operating characteristic (ROC) analysis. RESULTS: In differentiating low (GS 6) vs. intermediate (GS 7) risk PZ PCa, combination of (D, ß) provides the best performance with AUC of 0.829 with significance of p = 0.018 when compared to ADC (AUC of 0.655). In differentiating clinically significant (GS 6) vs. clinically significant (GS ≥ 7) PCa, combination of (D, ß, µ) provides highest AUC of 0.802 when compared to ADC (AUC of 0.671) with significance of p = 0.038. Stratification of intermediate (GS 7) and high (GS ≥ 8) risk PCa with FROC did not reach a significant difference when compared to ADC. CONCLUSION: Combination of FROC parameters shows greater performance than ADC at differentiating low vs. intermediate risk and clinically insignificant vs. significant prostate cancers in peripheral zone lesions. The FROC diffusion model holds promise as a quantitative imaging technique for non-invasive evaluation of PZ PCa.

Fabrication, Rheological, and Compositional Characterization of Thermoresponsive Hydrogel from Cornea.

Fabricating thermoresponsive hydrogels from decellularized tissues is a trending and promising approach to develop novel biomaterials for tissue engineering and therapeutic purposes. There are differences in the characteristics of the produced hydrogels related to the source tissue as well as the decellularization and solubilization protocols used. Detailed characterization of the hydrogels will support the efforts to optimize their application as biomaterials for tissue engineering and therapeutics. Here, we describe an optimized method for fabricating an in situ thermoresponsive hydrogel from decellularized porcine cornea extracellular matrix (COMatrix), and provide a detailed characterization of its structure, thermoresponsive rheological behavior (heat-induced sol-gel transition), as well as exploring its protein composition using proteomics. COMatrix forms a transparent gel (10-min time to gelation) after in situ curing with heat, characterized by alteration in light absorbance and rheological indexes. The rheological characterization of heat-formed COMatrix gel shows similar behavior to common biomaterials utilized in tissue engineering. The fibrillar structure of COMatrix gel was observed by scanning electron microscopy showing that the density of fibers attenuates in lower concentrations. Mass spectrometry-based proteomic analysis revealed that COMatrix hydrogel is rich in proteins with known regenerative properties such as lumican, keratocan, and laminins in addition to structural collagen proteins (Data is available via ProteomeXchange with identifier PXD020606). COMatrix hydrogel is a naturally driven biomaterial with favorable biomechanical properties and protein content with potential application as a therapeutic biomaterial in ocular regeneration and tissue engineering. Impact statement Fabrication and application of decellularized porcine corneal extracellular matrix is an emerging approach for corneal tissue engineering and regeneration. There are several protocols for decellularization of porcine cornea with various efficiencies. Here, we are presenting an optimized protocol for decellularization of porcine cornea followed by fabrication of a thermoresponsive hydrogel from the decellularized cornea matrix. Moreover, the fabricated hydrogel was rheologically and compositionally characterized as crucial features to be employed for further application of this hydrogel in corneal tissue engineering and regeneration.

Gene dosage manipulation alleviates manifestations of hereditary PAX6 haploinsufficiency in mice.

In autosomal dominant conditions with haploinsufficiency, a single functional allele cannot maintain sufficient dosage for normal function. We hypothesized that pharmacologic induction of the wild-type allele could lead to gene dosage compensation and mitigation of the disease manifestations. The paired box 6 (PAX6) gene is crucial in tissue development and maintenance particularly in eye, brain, and pancreas. Aniridia is a panocular condition with impaired eye development and limited vision due to PAX6 haploinsufficiency. To test our hypothesis, we performed a chemical screen and found mitogen-activated protein kinase kinase (MEK) inhibitors to induce PAX6 expression in normal and mutant corneal cells. Treatment of newborn Pax6-deficient mice (Pax6Sey-Neu/+ ) with topical or systemic MEK inhibitor PD0325901 led to increased corneal PAX6 expression, improved corneal morphology, reduced corneal opacity, and enhanced ocular function. These results suggest that induction of the wild-type allele by drug repurposing is a potential therapeutic strategy for haploinsufficiencies, which is not limited to specific mutations.

Uterine Carcinosarcoma: A Case Report and Literature Review.

Uterine carcinosarcomas are rare and extremely aggressive undifferentiated carcinomas which include both carcinomatous and sarcomatous elements. A 52-year-old female presented with heavy irregular menstrual bleeding for several years and new right elbow pain and swelling. Ultrasound and computed tomography showed a large uterine mass with regional and distant metastatic lymphadenopathy and suspicious findings of osseous metastasis to the right elbow. A biopsy confirmed uterine carcinosarcoma, and the patient underwent chemotherapy and then surgical resection of the uterine mass with palliative radiotherapy of the right elbow. The postoperative imaging showed new metastasis, and the patient was scheduled to start on immunotherapy. Considering the highly invasive nature of uterine carcinosarcomas, timely detection of this cancer using characteristic imaging and pathology findings is of extreme importance to improve the patient's survival.

The Effect of Mesenchymal Stem Cell Secretome on Corneal Endothelial Cell Preservation in an Oxidative Injury Model.

PURPOSE: To describe a reproducible oxidative injury model in ex vivo porcine corneas and to investigate the effects of corneal mesenchymal stem cell (Co-MSC) secretome and specific factors on the preservation of corneal endothelium after oxidative injury. METHODS: Porcine corneas underwent vital staining with trypan blue and alizarin red with different concentration and time points. Ex vivo porcine corneas were exposed (endothelial side) to varied concentrations of hydrogen peroxide. After injury, 3 groups of 5 corneas underwent treatment with secretome from either a wild-type (WT) murine Co-MSC, a pigment epithelium derived factor (PEDF) knock out (K/O) murine Co-MSC, or basal media for 4 hours at 37°C. The viability of the endothelium was evaluated using the optimized vital staining protocol. RESULTS: The optimal vital staining was achieved with 0.4% trypan blue for 60 seconds and 0.5% alizarin red for 90 seconds. The optimal oxidative injury (for consistency and level of damage) was obtained with 1% hydrogen peroxide for 15 seconds. Treatment with both WT Co-MSC and PEDF K/O Co-MSC secretome significantly reduced the endothelial damage compared with control (17.2% ± 10.0%, 33.5% ± 11.6%, and 68% ± 17%, respectively, P < 0.01). The WT Co-MSC secretome was significantly more effective compared with PEDF K/O Co-MSC secretome (P < 0.05). CONCLUSIONS: A reproducible model of vital staining and oxidative injury is described for studying porcine corneal endothelial survival. Our results demonstrate a beneficial role of a corneal MSC secretome in reducing oxidative damage to the corneal endothelium. In addition, it suggests a potential role for PEDF in this process.

Reproducible Derivation and Expansion of Corneal Mesenchymal Stromal Cells for Therapeutic Applications.

Purpose: A reproducible protocol for the production of corneal mesenchymal stem/stromal cells (cMSCs) is necessary for potential clinical applications. We aimed to describe successful generation and expansion of cMSCs using an explant method. Methods: Corneoscleral rims of human cadaveric eyes were divided into four pieces and used as explants to allow outgrowth of cMSCs (passage 0, or P0). The cells were subcultured at a 1:10 ratio until passage 5 (P5). The characteristics as well as therapeutic effects of expanded cMSCs were evaluated both in vitro, using a scratch assay, and in vivo using epithelial debridement and chemical injury mouse models. Results: All explants demonstrated outgrowth of cells by 7 days. Although the initial outgrowth included mixed mesenchymal and epithelial cells, by P1 only cMSCs remained. By subculturing each flask at a ratio of 1:10, the potential yield from each cornea was approximately 12 to 16 × 1010 P5 cells. P5 cMSCs demonstrated the cell surface markers of MSCs. The secretome of P5 cMSCs induced faster closure of wounds in an in vitro scratch assay. Subconjunctival injection of P5 cMSCs in mouse models of mechanical corneal epithelial debridement or ethanol injury led to significantly faster wound healing and decreased inflammation, relative to control. Conclusions: cMSCs can be reproducibly derived from human cadaveric corneas using an explant method and expanded with preservation of characteristics and corneal wound healing effects. Translational Relevance: The results of our study showed that cMSCs produced using this scheme can be potentially used for clinical applications.

A Concise Review and Required Precautions for COVID-19 Outbreak in Diagnostic and Interventional Radiology.

A global outbreak of a novel coronavirus (COVID-19) pneumonia began in December 2019 in Wuhan, China. The World Health Organization (WHO) announced a pandemic on March 11, 2020. The rapid rise in the case numbers and mortality led to the saturation of hospitals in many countries. COVID-19 patients usually present with fever, fatigue, dry cough, and dyspnea. Given the shortage of diagnostic kits in many countries and very high sensitivity of computed tomography (CT) for diagnosis of COVID-19 in clinically suspicious patients, the chest CT has been implemented among the primary initial methods of diagnosis before the confirmatory laboratory tests. This puts radiologists and radiology staff on the front line of this alarming pandemic. This report summarizes chest CT findings of COVID-19 patients to facilitate diagnosis and reviews a list of necessary precautions and safety measures for diagnostic and interventional radiology personnel. These precautionary plans are extremely important to avoid contamination of the health-care providers, as well as cross-contamination between patients.

10-year risk of cardiovascular disease and body mass index in association with the obesity paradox.

BACKGROUND: Some recent studies reported an inverse association between obesity and risk of cardiovascular diseases (CVD), heart failure related mortality rate, outcomes of myocardial infarction (MI), and the consequences of cardiovascular events interventions; this inverse association was named the obesity paradox. The present study was conducted with the aim to determine whether the obesity paradox will be detectable when the 10-year risk of CVD is estimated using CVD risk assessment tools. METHODS: The related data of 2910 subjects aged 40-74 years obtained in our cohort study that was carried out among 6140 subjects in Amol, in northern Iran, was included in this study. CVD risk assessment tools were used to estimate the 10-year risk of CVD. Obesity was evaluated using 4 indices, including waist circumference (WC), waist to height ratio (WHtR), waist to hip ratio (WHR), and body mass index (BMI). The receiver operating characteristic (ROC) curve analysis was utilized to evaluate the discriminatory power of obesity indices for 10-year risk of CVD. RESULTS: Categorizing the participants to with and without obesity according to BMI showed that a significantly higher proportion of men with obesity had a 10-year risk of CVD ≥ 7.5% and ≥ 10% according to American College of Cardiology/American Heart Association (ACC/AHA) and the Framingham approaches, respectively. A higher proportion of women without obesity had a 10-year risk of CVD ≥ 7.5% than women with obesity based on the ACC/AHA equation (28.54% vs. 24.15%; P = 0.0707). BMI had a non-significant AUC (< 0.5) according to the the ACC/AHA equation. CONCLUSION: BMI showed a weak and non-significant inverse association with 10-year risk of CVD estimated using pooled cohort equations of ACC/AHA in women. However, this result cannot directly provide enough evidence for the obesity paradox.

Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion.

Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.

New ex vivo model of corneal endothelial phacoemulsification injury and rescue therapy with mesenchymal stromal cell secretome.

PURPOSE: To develop a reproducible ex vivo model of corneal endothelial cell injury using phacoemulsification in porcine eyes and to evaluate the effects of mesenchymal stromal cell secretome in this injury model. SETTING: Department of Ophthalmology, University of Illinois at Chicago, Illinois, USA. DESIGN: Experimental study. METHODS: A corneal endothelial injury model was optimized using different powers and durations of ultrasound energy inside ex vivo porcine eyes. Conditioned media from corneal mesenchymal stem cells was collected under serum-free conditions from passages 4 to 6. Immediately after the phacoemulsification injury, the anterior chamber fluid was replaced with unconditioned media or conditioned media and incubated at 37°C for 4 hours. At the end, endothelial cell viability was evaluated using trypan blue staining and analyzed with ImageJ software. RESULTS: Using specific parameters (50% power for 30 seconds), phacoemulsification inside fresh porcine eyes led to a consistent level of endothelial cell injury. Incubation with corneal mesenchymal stromal cell-conditioned media after the injury significantly reduced endothelial cells loss compared with unconditioned media (mean 1.29% ± 0.91% [SD] and 5.33% ± 3.24%, respectively, P < .05). CONCLUSIONS: Phacoemulsification inside fresh porcine eyes provided a reproducible model to study endothelial cell injury. Treatment with corneal mesenchymal stromal cell secretome after injury appeared to significantly enhance the survival of corneal endothelial cells. This might provide a new strategy for preventing corneal endothelial cell loss after phacoemulsification or other endothelial injuries. Further in vivo studies are necessary to determine the therapeutic potential.

Effect of Human Corneal Mesenchymal Stromal Cell-derived Exosomes on Corneal Epithelial Wound Healing.

Purpose: Mesenchymal stromal cells (MSCs) have been used therapeutically to modulate inflammation and promote repair. Extracellular vesicles, including exosomes, have been identified as one of the important mediators. This study investigated the effect of human corneal MSC-derived exosomes on corneal epithelial wound healing. Methods: Corneal MSCs (cMSCs) were isolated from human cadaver corneas. The secretome was collected after 72 hours and exosomes were isolated using differential ultracentrifugation. Morphology and size of exosomes were examined by electron microscopy and dynamic light scattering. Expression of CD9, CD63, and CD81 by cMSC exosomes was evaluated by western blotting. Cellular uptake of exosomes was studied using calcein-stained exosomes. The effect of exosome on wound healing was measured in vitro using a scratch assay and in vivo after 2-mm epithelial debridement wounds in mice. Results: cMSC exosomes were morphologically round and main population ranged between 40 and 100 nm in diameter. They expressed CD9, CD63, and CD81, and did not express GM130, Calnexin, and Cytochrome-C. Stained cMSC exosomes were successfully taken up by human cMSCs, human corneal epithelial cells (HCECs), and human macrophages in vitro and by corneal epithelium in vivo. In scratch assay, after 16 hours, cMSC exosome treated HCECs had 30.1% ± 14% remaining wound area compared to 72.9% ± 8% in control (P < 0.005). In vivo, after 72 hours, cMSC exosome-treated corneas had 77.5% ± 3% corneal wound healing compared to 41.6% ± 7% in the control group (P < 0.05). Conclusions: Human cMSC exosomes can accelerate corneal epithelial wound healing, and thus, may provide a therapeutic approach for ocular surface injuries.