Postmortem Ultrastructural Analysis of the Retina from COVID-19 Deceased Patients.

PURPOSE: COVID-19 (coronavirus disease 2019) is an infectious disease caused by SARS-CoV-2, first reported in 2019 in Wuhan, China. Among the common complications is a pro-inflammatory and hypercoagulative response that compromises the vasculature among various organs. METHODS: In this report, we present the postmortem retinal findings of five patients observed by means of optical microscopy and transmission and scanning electron microscopy techniques. RESULTS: Clinical manifestations such as retinal hemorrhages and exacerbated inflammatory infiltrate, altered ultra structure with swollen mitochondria and pyknotic cells in both layers of the retina were observed in all analyzed eyes. CONCLUSION: Our data point to the fragility of this tissue in cases of severe COVID-19.

Clinical and anatomic correlates of objectively measured zonular dehiscence in 427 postmortem pseudophakic eyes.

OBJECTIVE: This study aims to objectively measure the degree of zonular dehiscence in postmortem eyes and to assess for clinical and anatomic correlates. DESIGN: Cross-sectional study. MATERIALS: Four hundred and twenty-seven postmortem pseudophakic human eyes. METHODS: Eyes were obtained from the Lions Gift of Sight Eye Bank. Microscope photographs were taken of the eyes in Miyake-Apple view, and region-of-interest analysis was performed using ImageJ to measure the area, circumference, and diameter of the capsular bag, the ciliary ring, and the capsulorhexis. Clinical and anatomic parameters were assessed using simple linear regression analysis and one-way analysis of variance with post hoc Bonferroni testing. Zonular dehiscence was measured via 2 surrogates: capsule area over ciliary ring area ratio (CCR) and capsule-ciliary ring decentration (CCD). Low CCR and high CCD indicate more zonular dehiscence. RESULTS: CCR was significantly inversely correlated with smaller capsulorhexi (p = 0.012), lower intraocular lens power (p < 0.00001), younger age at death (p = 0.00002), and a longer cataract-to-death time (p = 0.00786). CCR also was significantly lower in glaucomatous cases (p = 0.0291). CCD was significantly correlated with longer cataract-to-death time (p = 0.000864), larger ciliary ring area (p = 0.001), more posterior capsule opacification (p = 0.0234), and higher Soemmering's ring opacity (p = 0.0003). There was also significantly more decentration in male eyes than in female eyes (p = 0.00852). CONCLUSIONS: CCR and CCD are novel measures of zonular dehiscence in postmortem eyes, with many interesting correlates. An enlarged ciliary ring area is possibly associated with zonular dehiscence in pseudophakic eyes and may be a quantifiable surrogate in vivo.

Cemiplimab for metastatic squamous cell carcinoma of the orbit, periocular adnexa, and thigh.

The current case presentation highlights the potential of cemiplimab, a programmed cell death protein-1 (PD-1) inhibitor, as first-line treatment for periocular metastatic cutaneous squamous cell carcinoma (SCC) without requiring curative surgery or radiotherapy. A 64-year-old male presented with a progressing 4.5 × 3.0 cm left upper eyelid lesion initially diagnosed as psoriasis. Work-up revealed cutaneous SCC with tumor invasion into extraconal fat and lacrimal gland, and metastasis to the left parotid lymph node. The patient also presented with a suspicious lesion on his left medial thigh found to be a second primary on pathology. To avoid orbital exenteration and treat the multifocal disease, the patient was started on intravenous cemiplimab immunotherapy. Following six doses, repeated FGD-PET-CT revealed a complete response of the left eyelid lesion and residual low-grade hypermetabolic activity of the left medial thigh lesion. Biopsy confirmed chronic inflammation and fibrosis with no signs of malignancy. This unique case with dual primary cutaneous SCC provides support for cemiplimab in treating locally invasive periocular SCC, and potentially abrogating the need for highly morbid exenteration procedures to preserve binocular vision.

Histopathological analysis of residual lens cells in capsular opacities after cataract surgery using objective software.

Purpose: Remnant lens epithelial cells (LECs) within the capsular bag (CB) undergo epithelial-to-mesenchymal transition (EMT) and acquire a myofibroblast phenotype, depositing extracellular matrix (ECM) components, leading to posterior capsular opacification (PCO). This study histopathologically analyzes the LEC-to-myofibroblast transition and de novo ECM component deposition (i.e., smooth muscle actin (SMA) and fibronectin (FN) expression) and determines the intraocular lens (IOL) and patient factors associated with these changes. Methods: In total, 190 CBs with IOLs were removed from donor eyes. Digital images were obtained, and PCO was graded using published software (ADOS, Medical Parachute). Automated immunohistochemistry was performed using anti-SMA to detect EMT and anti-FN to document ECM remodeling. Slides were digitized and analyzed using the Positive Pixel Count v9 algorithm. Linear regression and Poisson regression were performed (P < 0.05). Results: SMA positive expression decreased as the time of IOL implantation increased (P < 0.0001). Positivity of SMA and FN demonstrated a positive correlation (P = 0.0002). Controlling for confounding factors in Poisson regression, hydrophobic and hydrophilic materials showed higher FN and SMA expression when compared to silicone material lenses (FN; P = 0.018; P < 0.0001, SMA; P = 0.001; P = 0.003, respectively). The square optic design had 29% higher SMA positivity compared to the opti-edge design (P = 0.042). One-piece haptic lenses had higher SMA expression compared to three-piece haptic (P = 0.042). A higher risk of expression of SMA and FN was seen in patients with a history of smoking, hypertension, and glaucoma (P < 0.05). Conclusion: This study demonstrated that SMA and FN expression is different according to IOL design and patient factors, thus indicating that LEC changes depend on lens biocompatibility. Therefore, by analyzing the histopathological composition of PCO by using LECs, further insight into the characteristics of IOLs that are important for biocompatibility can be ascertained.

Filtering blue light mitigates the deleterious effects induced by the oxidative stress in human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a major cause of blindness in elderly. It is characterized by the loss of central vision due to damaged retinal pigment epithelial (RPE) cells and photoreceptors. Blue Light (BL) exposure was proposed as a risk factor for AMD progression. We undertook this study to determine the effects of BL on the behaviour of RPE cells and their potential mitigation by BL-filtering intraocular lenses (IOL). Human RPE cells were exposed or not to BL, with the absence or presence of either a clear ultraviolet (UV)-filtering IOL (CIOL), or a yellow UV- and BL-filtering IOL (YIOL). Cells were analyzed for their oxidative stress by measuring the levels of reactive oxygen species (ROS), and their viability. BL exposure significantly increased the levels of both total cellular and mitochondrial ROS. While this increase was not affected by placing the CIOL in the BL beam, YIOL decreased the levels of both ROS reservoirs. Increased ROS production was accompanied by increased cell death which was similarly decreased when cells were protected with the YIOL. Pre-treatment of cells with N-acetylcycteine (NAC) abolished the increased cell death, suggesting that the effects of BL on cell viability were mainly due to increased levels of ROS. BL is deleterious to RPE cells due to increased oxidative stress and cell death. These effects were mitigated by filtering these radiations. The use of BL-filtering devices may represent a strategy to reduce these effects on RPE cells and delay the onset of AMD.

Ocular Disease in Mice Inoculated with Pork Heart Samples Infected with Toxoplasma gondii.

PURPOSE: We analyzed the frequency, viability, and genetic characteristics of T. gondii in pork heart samples. METHODS: Thirty-five fresh pork samples were purchased in a slaughterhouse in Erechim city. The DNA was extracted and qPCR was performed. T. gondii genotyping was performed using PCR-RFLP analysis. Positive samples were digested and inoculated in mice for viability analysis. RESULTS: Our results showed that T. gondii DNA was detected in 25.7% of the pork heart samples and genotyping revealed one new atypical strain. The viability analyses demonstrated that 40% of mice presented clinical signs of T. gondii infection. qPCR was positive in the lung, liver, and brain of mice that presented clinical signs of T. gondii infection. Also, the histopathology analysis showed retinal disorganization, retinal detachment, inflammatory cell infiltration, and fibrosis in the eyes analyzed. CONCLUSION: Our findings have shown that pork eat from southern Brazil may contain live T. gondii that could be associated with toxoplasmosis.

Uveal melanoma incidence trends in Canada: 1992-2010 vs. 2011-2017.

Background/aims: Uveal melanoma is the most common type of non-cutaneous melanoma and the most common ocular malignancy in the adult population, especially affecting Caucasians (98% of cases). Despite its low incidence rate, we have noted increasing incidence trends in recent years. Methods: We analyzed uveal melanoma incidence data using the Canadian Cancer Registry (CCR) for 2011-2017 years. The data was examined using the International Classification of Diseases for Oncology, Third Edition, codes for all uveal melanoma subtypes. The data for 2011-2017 was then compared to previously published work by our research group for uveal melanoma incidence in Canada between 1992 and 2010 using the same methodology. Results: Between 2011 and 2017, 1,215 patients were diagnosed with uveal melanoma, 49% of whom were females. The percentage distribution of uveal melanoma between the sexes was similar between 1992-2010 and 2011-2017, whereby of the 2,215 diagnoses of uveal melanoma in 1992-2010, 47.9% were females. The change in the incidence rate for this cancer has doubled between 1992-2010 and 2011-2017, from 0.074 to 0.15 cases per million individuals per year. Our study documents that the Canadian 2011-2017 age-standardized incidence rate (ASIR) for uveal melanoma against the World Health Organization (WHO) 2000-2025 world population standard was 5.09 cases per million individuals per year (95% confidence interval, 4.73-5.44), as compared with the 1992-2010 rate of 3.34 cases per million individuals per year (95% confidence interval, CI 3.20 to 3.47). Conclusion: This work demonstrates an ongoing, steady increase in uveal melanoma incidence in Canada in recent years.

Presumed SARS-CoV-2 Viral Particles in the Human Retina of Patients With COVID-19.

Importance: The presence of the SARS-CoV-2 virus in the retina of deceased patients with COVID-19 has been suggested through real-time reverse polymerase chain reaction and immunological methods to detect its main proteins. The eye has shown abnormalities associated with COVID-19 infection, and retinal changes were presumed to be associated with secondary microvascular and immunological changes. Objective: To demonstrate the presence of presumed SARS-CoV-2 viral particles and its relevant proteins in the eyes of patients with COVID-19. Design, Setting, and Participants: The retina from enucleated eyes of patients with confirmed COVID-19 infection were submitted to immunofluorescence and transmission electron microscopy processing at a hospital in São Paulo, Brazil, from June 23 to July 2, 2020. After obtaining written consent from the patients' families, enucleation was performed in patients deceased with confirmed SARS-CoV-2 infection. All patients were in the intensive care unit, received mechanical ventilation, and had severe pulmonary involvement by COVID-19. Main Outcomes and Measures: Presence of presumed SARS-CoV-2 viral particles by immunofluorescence and transmission electron microscopy processing. Results: Three patients who died of COVID-19 were analyzed. Two patients were men, and 1 was a woman. The age at death ranged from 69 to 78 years. Presumed S and N COVID-19 proteins were seen by immunofluorescence microscopy within endothelial cells close to the capillary flame and cells of the inner and the outer nuclear layers. At the perinuclear region of these cells, it was possible to observe by transmission electron microscopy double-membrane vacuoles that are consistent with the virus, presumably containing COVID-19 viral particles. Conclusions and Relevance: The present observations show presumed SARS-CoV-2 viral particles in various layers of the human retina, suggesting that they may be involved in some of the infection's ocular clinical manifestations.

Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. METHODS: We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCß4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). RESULTS: The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. CONCLUSIONS: We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.

Optical Coherence Tomography of Peri-Ocular Skin Cancers: An Optical Biopsy.

INTRODUCTION: Optical coherence tomography (OCT) imaging has been used as a diagnostic tool for retinal disease for several years, and OCT apparatuses are becoming increasingly powerful. However, OCT has yet to reach its full potential in ophthalmology clinics. Alike retinal layers, it has been shown that OCT is able to generate cross-sectional images of the skin and allows visualization of skin lesions in a histopathology-like manner. OBJECTIVE: We aim to validate OCT as an imaging modality for peri-ocular skin cancer. Through a series of cases, we highlight findings for 3 common eyelid malignancies: basal cell carcinoma, squamous cell carcinoma and sebaceous carcinoma. We propose an OCT image-based signature for basal cell carcinoma. METHODS: This is a prospective study. Fifty-eight lesions suspicious of malignancy from 57 patients were subjected to OCT imaging prior to the surgical excision of the lesion. OCT images were analysed and scored according to previously identified OCT features. Eight representative examples are presented, highlighting the OCT patterns for each malignancy side by side to its corresponding histopathological sections. RESULTS: Out of the 58 lesions analysed, 53 were malignant. A loss of the dermal-epidermal junction is observed in all malignant lesions. A strong link is observed between the presence of subepithelial hyporeflective nests on OCT and the diagnosis of basal cell carcinoma (present in 83% of cases). Conversely, lesions of epithelial origin such as squamous cell carcinoma are most often represented on OCT by acanthosis. Two supplementary cases, one basal cell carcinoma and one sebaceous carcinoma, are provided to illustrate how OCT imaging is a valuable tool in cases where clinical observations may be unusual. CONCLUSIONS: We provide evidence supporting the use of OCT for the evaluation of peri-ocular cancers. OCT enables visualization of the skin layers in vivo, before biopsy. Our results show that certain OCT features can contribute to include or exclude a diagnosis of basal cell carcinoma. By integrating this non-invasive imaging methodology into the routine assessment of peri-ocular skin lesions, especially in health care centres where access to specialists is limited, OCT imaging can increase clinical precision, reduce delays in patient referral and enhance patient care.

Intratumoral Heterogeneity in Uveal Melanoma.

Tumor biopsies in uveal melanoma (UM) serve mainly the purpose of prognostication and assessment of individual metastatic risk, but can be used for diagnosis in selected cases. The importance of precise information is paramount for selecting adequate surveillance protocols, patient counseling, and optimization of treatment strategies. However, intratumoral heterogeneity and sample representativity are major concerns and can interfere with the correct prediction of the patient's prognosis. We report a series of cases of UM with distinct morphologically identifiable areas, highlighting the differences in clinical behavior, as well as histopathological and genetic features.

Histopathological Variability and Concomitant Lesions in Pterygium in a Large Case Series.

Pterygium is a common lesion consisting of fleshy conjunctival growth extending towards the cornea. There is no documented risk of malignant transformation; however, concomitant disease is not rare, and its link to sunlight exposure indicates a risk of other malignancies. The purpose of our study is to describe histopathological features of resected pterygiums and to recognize patients at risk of other conjunctival diseases. One hundred and forty-nine formalin-fixed and paraffin-embedded pterygium samples were subjected to histopathological analysis. Histological H&E sections were obtained and digitalized using a Zeiss Axio Scan.Z1 slide scanner. Thirteen predefined morphological features were used to record histopathological changes in the epithelium and substantia propria. Neovascularization was observed in 54% of the samples. Sun damage, comprising solar elastosis and stromal plaque, was present in 81% of the samples. Variation in epithelial thickness was the most common change, with acanthosis and atrophy being observed in 62% and 26% of the samples, respectively. In our series, 21% (31/149) of pterygiums showed mild to moderate dysplasia, a finding that may be associated to ocular surface squamous neoplasia (OSSN). Moreover, 32% (47/149) of the cases showed melanocytic hyperplasia, which could represent primary acquired melanosis (PAM). There is a positive correlation between dysplasia and chronic inflammation (p=0.012) and an inverse correlation with epithelial atrophy (p=0.001) and neovascularization (p=0.05). Similarly, a positive correlation is observed between goblet cell hyperplasia and melanocytic hyperplasia (p=0.02). Our findings show that pterygiums harbour histological features that may be suggestive of OSSN or PAM in 53% of our patients. Whilst being on the benign side of the spectrum, these two entities are known for their potential progression to malignancy. A recommendation is made for all surgically excised pterygiums to be sent for histopathological diagnosis, and clear guidelines for reporting of these lesions should be established. Associated histopathological findings suggestive of other concomitant diseases should be identified to insure adequate follow-up of these patients.

Histopathological changes in the anterior segment with anterior and posterior chamber intraocular lens.

OBJECTIVE: Patients have shown a lowering of intraocular pressure (IOP) after cataract surgery. Histopathology studies have reported trabecular meshwork (TM) changes in pseudophakic eyes with posterior chamber intraocular lens (PCIOL) and have eluded to the mechanisms for IOP decrease. Unlike PCIOLs, TM histopathology changes after implantation of an anterior chamber intraocular lens (ACIOL) have not been studied, to our knowledge. Therefore, this study aims to examine the histopathological changes in both the TM and corneal endothelium among donor eyes with ACIOL, PCIOL, and phakic eyes. METHODS: Forty fixed postmortem donor eyes were obtained, sectioned, and embedded. Slides were stained with Masson's trichrome and CD31 vascular endothelial antibody, and further digitalized. Customized Medical Parachute TMAN software quantified the cellular components, the trabecular extracellular matrix (ECM), ECM fibrosis, and trabecular area. Schlemm's canal and corneal endothelium were quantified across the ACIOL, PCIOL, and phakic groups. RESULTS: Cellular area component of the TM was lower in the ACIOLs and PCIOLs than in phakic eyes, but statistically significant only between PCIOL and phakic eyes (p = 0.0023). ECM area component, TM fibrosis score and TM lamellae area, ciliary process fibrosis, and CD31 expression in Schlemm's canal showed no differences (p = 0.40, 0.99, 0.10, 0.83, 0.45). Significantly lower corneal endothelial cells were seen in ACIOLs compared with both PCIOLs and phakic eyes (p = 0.0002). CONCLUSIONS: ACIOLs and PCIOLs in our sample group showed that there is loss of cellular components in the TM compared with the phakic eyes, with PCIOLs displaying the least amount of TM cells statistically, in this cohort. The ACIOLs led to a greater loss of corneal endothelial cells than both PCIOLs and phakic eyes after cataract surgery. The endothelial cells in Schlemm's canal did not seem to be affected by the IOL placements. Therefore, this study illustrates that there are histopathological differences seen with the placements of ACIOLs in both TM and cornea.

The chemotactic properties of various topical brimonidine tartrate ophthalmic preparations.

BACKGROUND: The study aimed to evaluate and compare the leukocyte chemotactic activities of various brimonidine tartrate (BT) eye drop formulations. METHODS: A 96-well dot-blot platet using a Boyden-style well was used to study the chemotactic effects of BT ophthalmic preparations. A modification was made to create blind wells where the tested agents were placed. Leukocytes were isolated from the peripheral blood of healthy volunteers. As positive controls, we used diluted drugs, benzalkonium chloride solution (BAK), zymosan-activated serum, and formyl-methionine-leucine-phenylalanine peptides. The negative control in our study was a phosphate-buffered saline solution. For each experimental condition, we measured leukocyte migration through a Millipore membrane. The differences in the mean migration distance between groups were compared using the analysis of variance (ANOVA). RESULTS: The measured migration distances (in µm ± SD) were 62.14 ± 3.71 for BT 0.2% with BAK (Alcon Laboratories Inc.); 63.61 ± 3.81 for BT 0.2% with BAK (Allergan Inc); 40.36 ± 3.17 for BT 0.15% without BAK; and 41.02 ± 2.17 for BAK alone. The negative controls showed no chemotactic activity, while the positive controls showed the highest neutrophil migration of all experimental conditions. The differences between BT 0.15% without BAK and the other commercial formulations were statistically significant. CONCLUSION: Commercial ophthalmic preparations of BT 0.2% with BAK 0.005% had higher chemotactic properties than the alternative of a lower concentration of BT and without the preservative BAK. Therefore, the latter should be considered for patients with glaucoma or ocular hypertension in order to minimize iatrogenic ocular inflammation.

Epidemiology of invasive ocular surface squamous neoplasia in Canada during 1992-2010.

BACKGROUND: Ocular surface squamous neoplasia (OSSN) is the most common non-pigmented ocular surface malignancy. It is classified as invasive OSNN (IOSSN) when the underlying stroma are infiltrated by dysplastic squamous epithelial cells through the basement membrane. Here, we present the descriptive epidemiology and geographical distribution of IOSSN in Canada. METHODS: We determined the incidence and geographical distribution of IOSSN cases diagnosed between 1992 and 2010 using two independent population-based cancer registries: the Canadian Cancer Registry and Le Registre Québécois du Cancer. RESULTS: The mean annual age-standardised incidence rate (WHO 2000-2025) of IOSSN for 1992-2010 was 0.45 cases per million individuals per year with an average annual percent increase in incidence of 4.5%. IOSSN localisation to the conjunctiva was documented in at least 57% of the reported cases. IOSSN exhibited a male predilection ratio of 3.3:1.0 with a mean age at diagnosis of 69 years. Incidence rates of IOSSN across Canadian provinces and cities showed no significant differences from the crude national average. CONCLUSIONS: Our results, particularly concerning IOSSN patient age and male predilection, corroborate with data reported from the USA. Additional studies are needed to determine whether the observed increase in incidence rate over the study period (1992-2010) is significant.

Acetylsalicylic Acid Exerts Potent Antitumor and Antiangiogenic Effects in Cutaneous and Uveal Melanoma Cell Lines.

INTRODUCTION: Acetylsalicylic acid (ASA) has been investigated for a potential anticancer role in several cancers, such as colorectal, ovarian, and endometrial cancer. Moreover, ASA has been shown to abrogate various processes that contribute to tumor growth and progression. OBJECTIVE: The aim of this study was to evaluate the effects of ASA on cutaneous melanoma (CM) and uveal melanoma (UM). METHODS: Human CM and UM cells were treated with 5 mM ASA and assessed for changes in cellular functions. Antiangiogenic effects of ASA were determined using an ELISA-based assay for 10 proangiogenic cytokines, and then validated by Western blot. Finally, proteomic analysis of ASA-treated cells was performed to elucidate the changes that may be responsible for ASA-mediated effects in melanoma cells. RESULTS: Treatment with ASA significantly inhibited the proliferation, invasion, and migration capabilities, and caused a significant decrease in angiogenin and PIGF secretion in both CM and UM. Mass spectrometry revealed 179 protein changes associated with ASA in the CM and UM cell lines. CONCLUSIONS: These results suggest that ASA may be effective as an adjuvant therapy in metastatic CM and UM. Future studies are needed to determine the regulating targets that are responsible for the antitumor effects of ASA.