A 1H NMR metabolomic approach for the estimation of the time since death using aqueous humour: an animal model.

INTRODUCTION: The estimation of the time since death, or post-mortem interval (PMI), still remains a main conundrum in forensic science. Several approaches have been so far proposed from either a qualitative or a quantitative point of view, but they still lack reliability and robustness. Recently, metabolomics has shown to be a potential tool to investigate the time-related post-mortem metabolite modifications in animal models. OBJECTIVES: Here we propose, for the first time, the use of a 1H NMR metabolomic approach for the estimation of PMI from aqueous humour (AH) in an ovine model. METHODS: AH samples were collected at different times after death (from 118 to 1429 min). 1H NMR experiments were performed and spectral data analysed by multivariate statistical tools. RESULTS: A multivariate calibration model was built to estimate PMI on the basis of the metabolite content of the samples. The model was validated with an independent test set, obtaining a prediction error of 59 min for PMI < 500 min, 104 min for PMI from 500 to 1000 min, and 118 min for PMI > 1000 min. Moreover, the metabolomic approach suggested a picture of the mechanisms underlying the post-mortem biological modifications, highlighting the role played by taurine, choline, and succinate. CONCLUSION: The time-related modifications of the 1H NMR AH metabolomic profile seem to be encouraging in addressing the issue of a reproducible and robust model to be employed for the estimation of the time since death.

Optical coherence tomography in forensic sciences: a review of the literature.

Optical coherence tomography (OCT) is an interferometric imaging technique that has revolutionized clinical ophthalmology since the first half of the 1990's. Despite this approach being successfully employed in ophthalmology and having great potential in forensic cases, its use in different forensic fields appears to be quite limited. In this review we reviewed the scientific literature regarding the application of OCT in forensic science and legal medicine from 1995 to 2019. Our research showed the usefulness of this approach for the study of coronary injuries, postmortem ocular changes, forensic entomology, and several other applications of specific forensic interest (the study of blood stains, fingerprints, and hair bulbs for personal identification, as well as the study of materials found in the crime scene for comparation, or anti-fraud investigation). The creation of specific 'ad hoc' devices and a better knowledge of this type of technology by pathologists will be a fundamental step to continue to develop the use of OCT forensic fields.

Ocular surface and respiratory tract damages from occupational, sub-chronic exposure to fluorspar: case report and other considerations.

OBJECTIVES: To report the ocular surface and respiratory tract damages due to a sub-chronic (3 months), occupational exposure to fluorspar dust in case of inconsistent use of personal protective equipment. METHODS: A 50-year-old man was referred to our clinic with bilateral conjunctival injection, ocular foreign body sensation and symptoms of ocular discomfort. He reported having 3 days before an urgent hospital admission for dyspnoea with odynophagia, hyposmia, nausea, vomiting, headache and asthenia. Otorhinolaryngological examination revealed a severe hypertrophic rhinopharyngitis and a significant decrease in overall sensitivity to olfactory stimuli. General anamnesis was negative for previous diseases or medication use. RESULTS: Ocular examination revealed a diffuse injection of the ocular surface with some areas of conjunctival and limbal ischaemia, a best-corrected visual acuity of 20/25 in both eyes, an intraocular pressure of 23 and 21 mmHg, respectively, in the right and in the left eyes, and a normal fundus oculi. Assessment of the tear film and ocular surface showed the presence of dry eye disease. Based on medical history and clinical tests, initial diagnostic hypothesis was ocular surface burn due to chemical exposure, i.e. to fluorspar. Therefore, anterior segment fluorescein angiography was performed to confirm the extension of conjunctival and limbal ischaemia. Thus, ocular ischaemia was detected in approximately 10 clock hours of limbus and in 50% of conjunctiva. CONCLUSIONS: The present work remarks the importance of using protective equipment for preventing ocular and respiratory tract damages in workers occupationally exposed to the 'acid-grade fluorite'.

Morphological analysis of corneal findings modifications after death: A preliminary OCT study on an animal model.

The aim of this work was to describe, for the first time, the morphological modifications, in a three-dimensional mode, of the central cornea at different intervals since death. The study design involved the analysis of 30 eyes (15 heads) of female, adult sheep (>2 years) sacrificed at a local slaughterhouse. The eyes, after animal decapitation, were examined in situ, without enucleation. Ocular globes were stored at well-known temperature (within a range of 12-22 °C) and humidity (within a range of 50-60%). The instrumental analysis was executed using a portable spectral-domain OCT (SD-OCT) system (iVue SD-OCT, Optovue Inc, Fremont, CA) calibrated to the corneal mode. OCT imaging was performed at different time-points since death. Pachymetric map, morphological and ultrastructural analysis (epithelium, stroma, and endothelium), were performed for each time-point. After an initial thinning of tissues and an enhancement of epithelial reflectivity, stromal thickness increased from the 2nd up to the 6th hour. Subsequently, a new trend incorneal thinning was observed in association with the appearance ofone or more demarcation lines between the anterior andposterior stroma. After the 12th hour, a recurrence of corneal swelling was detected in association with thedelamination of stromal tissue. Since the 24th hour, the epithelium disappeared in 50% of cases and the anterior chamberdepth progressively decreased. At the 48th hour, various ocular structures showed the onset of putrefaction processes, such as theappearance of hyper-reflective dots in anterior chamber, iridocorneal contact, and the massive vacuolization of theposterior stroma until the total delamination. The portable OCT system is a useful approach for in situ postmortem corneal examination, and it may be potentially applied for the selection of donor cornea in transplantology and for the determination of post-mortem intervals in forensic medicine.

An uncommon case of intermittent Pourfour du Petit Syndrome associated with acute angle-closure glaucoma successfully treated by laser iridotomy.

PURPOSE: To describe a case of acute angle-closure glaucoma secondary to intermittent mydriasis related to Pourfour du Petit Syndrome caused by tracheal deviation. MATERIALS AND METHODS: A 70-year-old Caucasian woman visited the Emergency Room of the University Eye Clinic complaining of blurring of vision and difficulty to move superior eyelid in her right eye. Examination revealed reactive mydriasis, and upper lid retraction on the right side. The rest of the ophthalmological examination was normal, and a cranial computed tomography (CT) did not identify any abnormalities. A cervical CT showed the presence of an accentuated lateral right convex deviation of the trachea, attributable to a fibrothorax. A right Pourfour du Petit syndrome was suspected. Although the mydriasis had in the meantime vanished, the patient was admitted to the Neurological Clinic. RESULTS: Five days later she suffered acute pain in her right eye. Ophthalmological examination of the right eye revealed conjunctival hyperemia, marked corneal edema, reduced depth of anterior chamber, permanent mydriasis. As assessed by Goldmann applanation tonometry, intraocular pressure (IOP) was 48 mm Hg. Fundus examination was normal in both eyes. Gonioscopy revealed angle closure in all quadrants. Slit lamp examination of the contralateral eye was normal; IOP was 10 mm Hg. After hypotensive medical therapy, iridotomy with YAG laser was performed. Thereafter, IOP stabilized at 12 mm Hg. CONCLUSIONS: This is the first report in the literature of a case of acute angle-closure glaucoma secondary to mydriasis related to Pourfour du Petit Syndrome caused by tracheal deviation.

The Influence of Eyelid Position and Environmental Conditions on the Corneal Changes in Early Postmortem Interval: A Prospective, Multicentric OCT Study.

In the current study, using portable optical coherence tomography, we evaluated 46 corneas of 23 individuals in a multicenter setting during the first 17 h after death. Twenty-three eyes were kept open, and twenty three were kept closed. Furthermore, the experiment was carried out for 12 samples in summer and 11 in winter. Our data show that postmortem corneal alterations largely depend on the phenomena of dehydration (in particular in open eyes) and swelling of the stroma in closed eyes, probably due in the first phase to hypoxia/anoxia and subsequently to the passage by osmosis of the aqueous humor from the anterior chamber to the corneal tissue. Our findings could have significant repercussions in forensic pathology for estimating the postmortem interval and transplantation to optimize the conservation of the tissue before the explant.

The "Quarantine Dry Eye": The Lockdown for Coronavirus Disease 2019 and Its Implications for Ocular Surface Health.

The pandemic of coronavirus disease 2019 (COVID-19) has led many countries of the world to impose a series of containment measures such as lockdowns (mass quarantines), curfews or similar restrictions (eg, stay-at-home orders, or shelter-in-place orders). All these restrictions were established in order to limit spread of COVID-19. Thus, approximately 3.9 billion people worldwide were under lockdown by early April 2020. During this time (home confinement), some solutions have been proposed by experts to improve work and school productivity, including smart working and online school lessons. However, many of the restrictive measures are likely to act as predisposing factors for dry eye disease (DED), directly or related to sick building syndrome (SBS). Herein, we discuss the implications of quarantine measures on eye health, in particular on DED associated with SBS, and introduce some potential preventive strategies for lockdown-related ocular surface disorders. Several risk factors are implicated in their pathogenesis, including environmental changes (eg, air quality) and modifications in personal behaviors (eg, the abuse of digital devices, malnutrition, and sleep/psychiatric disorders). Considering a number of predisposing factors for DED, it is possible to state that patients under lockdown are at risk of ocular surface alterations. Accordingly, the COVID-19 pandemic era is expected to determine an increase in dry eye patients all around the world (a new phenomenon that we propose to name the "quarantine dry eye") in the event that the restrictive measures will be recursively extended over time.

Dual Corneal-Graft Rejection after mRNA Vaccine (BNT162b2) for COVID-19 during the First Six Months of Follow-Up: Case Report, State of the Art and Ethical Concerns.

Present mass vaccination against Coronavirus Disease-19 (COVID-19) is the most widely used health policy and the most promising approach to curb the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic globally. However, new side effects are emerging from the mass vaccination not described during the experimental stages. In the present study, we discuss a case of acute corneal graft rejection, which has occurred 25 years after transplantation and 13 days after the administration of the BNT162b2 vaccine (Comirnaty, BioNTech/Pfizer), which was followed-up for a period of six months. In this period, the corneal inflammation appeared twice but was successfully managed with topical therapy and supplementation of Vitamin D. A risk of corneal graft rejection must be included in the list of potential vaccine complications, in order to inform the transplanted patient to undergo a preliminary and a follow-up ocular examination, and eventually to include corneal graft in the list of contraindications to vaccination.

Postmortem Ocular Findings in the Optical Coherence Tomography Era: A Proof of Concept Study Based on Six Forensic Cases.

Postmortem analysis of the ocular globe is an important topic for forensic pathology and transplantology. Although crucial elements may be gathered from examining cadaveric eyes, the latter do not routinely undergo in-depth analysis. The paucity of quantitative and objective data that are obtainable using current, invasive necroscopic techniques is the main reason for the limited interest in this highly specialized procedure. The aim of the current study is to describe and to object for the first time the postmortem ocular changes by mean of portable optical coherence tomography for evaluating ocular tissues postmortem. The design involved the postmortem analysis (in situ, and without enucleation) of 12 eyes by portable spectral-domain Optical Coherence Tomography. The scans were performed, in corneal, retinal and angle modality at different intervals: <6 h, 6th, 12th, and 24th hour and after autopsy (25th-72nd hour). The morphological changes in the cornea, sclera, vitreous humor and aqueous humor were easy to explore and objectify in these tissues in first 72 h postmortem. On the other hand, the "in situ" observation of the retina was difficult due to the opacification of the lenses in the first 24 h after death.

Global Spread of Coronavirus Disease 2019 and Malaria: An Epidemiological Paradox in the Early Stage of A Pandemic.

In the current work, we discovered and analyzed the epidemiological paradox between coronavirus disease 2019 (COVID-19) and malaria in the initial phase of the ongoing pandemic. From the analysis of distribution data, the endemic presence of malaria seems to protect some populations from COVID-19 outbreak, particularly in the least developed countries. In this sense, molecular and genetic variations associated with malaria (e.g., in ACE2) might play a protective role against coronavirus infection. Moreover, the mechanism of action of some antimalarial drugs, e.g., the antiviral function, suggests their potential role in the chemoprophylaxis of coronavirus epidemics, despite possible adverse effects (e.g., retinal toxicity). All these data provide important insights to understand the spreading mechanisms of COVID-19, and to direct scientific research toward the study of some currently available medications.

Safety Recommendations and Medical Liability in Ocular Surgery during the COVID-19 Pandemic: An Unsolved Dilemma.

Ocular surgery is one of the most performed medical procedures in the world. Its limitation or suspension, recursively extended over time, could be associated with a significant increase in the number of blind people worldwide. Indeed, cataract causes more than half of all cases of visual impairment in those countries with limited availability of means for performing eye operations (e.g., Africa or India). In this scenario, the pandemic of coronavirus disease 2019 (COVID-19) quickly resulted in the suspension or sharp reduction of various ophthalmic activities considered non-urgent, including lens replacement surgery or some intraocular injections. Despite the imperative need to continuously practice eye operations to avoid the abovementioned problems, there are currently little-shared and vague recommendations among the various countries on safety in operating rooms (for health care workers and patients) and poor legal protection for surgeons (potentially transmitting the COVID-19 infectious agent). Herein, we individuated and discussed some critical points in safety recommendations and medical liability. A paradigm shift for ocular surgery during the COVID-19 era is now mandatory. While telemedicine has been able to solve some problems in clinical ophthalmology, the lack of adequate health and legal protection for surgeons and patients may result in an excessive reduction in the volume of surgical interventions during a pandemic era and the immediately following period, thus determining inability to ensure health care to all patients.

The Ocular Surface and the Coronavirus Disease 2019: Does a Dual 'Ocular Route' Exist?

Coronavirus disease 2019 (COVID-19) is an important health problem that was defined as a pandemic by the World Health Organization on 11 March 2020. Although great concern has been expressed about COVID-19 infection acquired through ocular transmission, its underlying mechanism has not currently been clarified. In the current work, we analyzed and elucidated the two main elements that should be taken into account to understand the "ocular route", both from a clinical and molecular point of view. They are represented by the dynamism of the ocular surface system (e.g., the tear film turnover) and the distribution of ACE2 receptors and TMPRSS2 protein. Although it seems, at the moment, that there is a low risk of coronavirus spreading through tears, it may survive for a long time or replicate in the conjunctiva, even in absence of conjunctivitis signs, indicating that eye protection (e.g., protective goggles alone or in association with face shield) is advisable to prevent contamination from external droplets and aerosol.

The architecture of corneal stromal striae on optical coherence tomography and histology in an animal model and in humans.

The purpose of this study was to use a portable optical coherence tomography (OCT) for characterization of corneal stromal striae (CSS) in an ovine animal model and human corneas with histological correlation, in order to evaluate their architectural pattern by image analysis. Forty-six eyes from female adult sheep (older than 2 years), and 12 human corneas, were included in our study. The eyes were examined in situ by a portable OCT, without enucleation. All OCT scans were performed immediately after death, and then the eyes were delivered to a qualified histology laboratory. In the ovine animal model, CSS were detected with OCT in 89.1% (41/46) of individual scans and in 93.4% (43/46) of histological slices. In human corneas, CSS were found in 58.3% (7/12) of cases. In both corneal types, CSS appeared as "V"- or "X"-shaped structures, with very similar angle values of 70.8° ± 4° on OCT images and 71° ± 4° on histological slices (p ≤ 0.01). Data analysis demonstrated an excellent degree of reproducibility and inter-rater reliability of measurements (p < 0.001). The present study demonstrated that by using a portable OCT device, CSS can be visualized in ovine and human corneas. This finding suggests their generalized presence in various mammals. The frequent observation, close to 60%, of such collagen texture in the corneal stroma, similar to a 'truss bridge' design, permits to presume that it plays an important structural role, aimed to distribute tensile and compressive forces in various directions, conferring resilience properties to the cornea.

COVID-19 and Italian Healthcare Workers From the Initial Sacrifice to the mRNA Vaccine: Pandemic Chrono-History, Epidemiological Data, Ethical Dilemmas, and Future Challenges.

On March 11, 2020, the World Health Organization (WHO) declared the coronavirus disease 2019 (COVID-19) outbreak a pandemic. Simultaneously, in Italy, in which the first case had occurred on February 18, the rigid phase of the lockdown began. The country has attracted worldwide attention, becoming at the same time a field of study both concerning the spread of the pandemic and advanced assessments of the effectiveness of political, public health, and therapeutic measures. The protagonists of the Italian crisis were the healthcare workers (HCWs) who were exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without having any perception of what they were facing, courageously contributing to the containment of the epidemic to be defined by the media as "heroes." However, in the first phase of the pandemic (March-May 2020), the price that the Italian Public Health System had to pay both in terms of the number of positive virus cases and deaths among the HCWs was beyond and represented a peculiarity compared to what happened in other countries. In the current study, after a summary of the evolution of the pandemic in Italy, we offer an analysis of the statistical data concerning contagions and deaths among healthcare workers (physicians in particular). In conclusion, we describe the critical issues that still need to be resolved and the future challenges facing healthcare workers and the general population.

A Panel of Broad-Spectrum Antivirals in Topical Ophthalmic Medications from the Drug Repurposing Approach during and after the Coronavirus Disease 2019 Era.

The coronavirus disease 2019 (COVID-19) represents a global concern of public health caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Its clinical manifestations are characterized by a heterogeneous group of symptoms and pictures (ranging from asymptomatic to lethal courses). The prevalence of conjunctivitis in patients with COVID-19 is at present controversial. Although it has been reported that only 0.9% developed signs of conjunctivitis, other report indicates that up to 31.6% of hospitalized patients had conjunctivitis. Considering the widespread use of topical ophthalmic medications (e.g., eye drops) by the general population, for various reasons (e.g., artificial tears, anti-glaucoma medications, topical antibiotics, etc.), the existence of their side effects as antiviral action should be investigated in-depth because it could possibly explain the aforementioned controversial data and represent a potential antiviral treatment for SARS-CoV-2 replication/diffusion on the ocular surface. Here, we discuss and elucidate the antiviral side effect of many eye drops and ophthalmic ointments commonly used for others purposes, thus showing that these secondary effects (not to be confused with the 'adverse effects') might be of primary importance in a number of viral infections (e.g., those for which there is no validated treatment protocol), according to a drug repurposing approach. Some active ingredients or excipients described here have activity against other types of viruses, thus suggesting potential broad-spectrum applications.

Repeatability and reproducibility of post-mortem central corneal thickness measurements using a portable optical coherence tomography system in humans: a prospective multicenter study.

To assess the repeatability and reproducibility of post-mortem central corneal thickness (CCT) measurements made by the portable iVue spectra-domain (SD) optical coherence tomography OCT (Optovue Inc, Fremont, CA) system in humans, and to prospectively establish the time-course of CCT after death. In a prospective multicenter setting, CCT measurements were obtained from 58 human eyes at the following 16 time-points after death: immediately (within 2 h), and at each hour by the next 17 h. The range of CCT values for each subject was determined and longitudinal data were used to illustrate the variation in open and close eye mode. All measurements were made by two independent and well-trained examiners for session. Main outcome measures were intraclass correlation coefficients (ICC), repeatability and reproducibility coefficients, and coefficients of variation of the average central (0-2 mm). Overall, a total of 5,568 OCT measurements were performed by examiners. The repeatability coefficient varied from 0.3 to 1.7% and the reproducibility coefficient varied from 0.3 to 1.6% throughout the entire experimental time frame. Furthermore, the values of the different ICCs were also high during the different postmortem intervals, thus demonstrating the excellent repeatability and reproducibility of the present OCT approach. When CCT measurements were analyzed longitudinally, corneal thickness showed different behavior based on the open or close eye mode. The present study demonstrates that portable OCT imaging can be reliably used for corneal pachymetric measurements in supine subjects and during the post mortem period, i.e. without visual fixation and normal physiology/architecture of examined tissues.

Fourier-Domain OCT Imaging of the Ocular Surface and Tear Film Dynamics: A Review of the State of the Art and an Integrative Model of the Tear Behavior During the Inter-Blink Period and Visual Fixation.

In the last few decades, the ocular surface and the tear film have been noninvasively investigated in vivo, in a three-dimensional, high resolution, and real-time mode, by optical coherence tomography (OCT). Recently, OCT technology has made great strides in improving the acquisition speed and image resolution, thus increasing its impact in daily clinical practice and in the research setting. All these results have been achieved because of a transition from traditional time-domain (TD) to Fourier-domain (FD) technology. FD-OCT devices include a spectrometer in the receiver that analyzes the spectrum of reflected light on the retina or ocular surface and transforms it into information about the depth of the structures according to the Fourier principle. In this review, we summarize and provide the state-of-the-art in FD-OCT imaging of the ocular surface system, addressing specific aspects such as tear film dynamics and epithelial changes under physiologic and pathologic conditions. A theory on the dynamic nature of the tear film has been developed to explain the variations within the individual compartments. Moreover, an integrative model of tear film behavior during the inter-blink period and visual fixation is proposed.

The Bull's Eye Pattern of the Tear Film in Humans during Visual Fixation on En-Face Optical Coherence Tomography.

The aim of the study was to define and characterize the optical behavior of the tear film during visual fixation in humans on en-face optical coherence tomography (OCT). We included 20 healthy participants, 60% female, aged from 25 to 42 years (33.05 ± 4.97 [mean ± SD]) and ten patients with severe dry eye, 50% female, aged from 26 to 42 years (33.7 ± 5.31). To perform high-resolution tear film imaging, participants were asked to gaze at the internal fixation point in the spectral-domain anterior segment OCT device, and meanwhile scanning session was executed at the following time-points after blinking: at the 2nd, 3rd, 4th, 5th, and 6th second. After one hour, OCT imaging was repeated (second session) by a different operator masked to the study to verify the reliability of results. During each measuring session, a pulse oximetry was used for continuously measuring the heart rate and oxygen saturation (SpO2%). A preliminary experiment was also performed to test the absence of geometric patterns from the anterior surface of a motionless artificial eye. OCT imaging showed a motionless, stable anterior surface of the artificial eye and in dry eye patients. Conversely, in the healthy participants of the study, a bull's eye pattern of the tear film was detected by OCT at the 2nd, 3rd, 4th, 5th, and 6th second after blinking, respectively, in 45%, 60%, 45%, 60%, and 40% of OCT scans during the first session, and in 35%, 65%, 65%, 60%, and 35% of cases in the second session. Overall, a total of 200 OCT scans were performed in normal human population. A significant correlation was found between the novel tear film pattern and heart rate during the first and the second session (p < 0.01) in healthy eyes. Conversely, no correlation was revealed with SpO2%. Intraclass correlation (ICC) analysis for OCT imaging of the tear film revealed a statistically significant reproducibility of the results (ICC = 0.838; p < 0.01), indicating the high level of reliability of the method, independently of heart rate and SpO2% variables. There exists a novel, geometric pattern of the tear film during visual fixation detectable by en-face OCT, which is mainly evident as heart rate increases. Its discovery implies in turn the presence of a specific vibration (or imperceptible motion) of the tear film that, at present, is not recognized and corrected by the OCT software (in image postprocessing) unlike other eyeball movements.

A study of refractory cases of persistent epithelial defects associated with dry eye syndrome and recurrent corneal erosions successfully treated with cyclosporine A 0.05% eye drops.

Background: Effective and tolerable therapeutic strategies for patients with refractory persistent epithelial defects (PEDs) are limited and generally provide poor outcomes. This retrospective case review describes four refractory cases of PEDs associated with recurrent corneal erosions (RCEs) and dry eye disease (DED), which were successfully treated with cyclosporine eye drops. Methods: Patients were treated with cyclosporine A 0.05% eye drops twice a day for at least 12 months. At enrolling time, each patient was asked to suspend topical steroids or other eye drops used for ocular surface abnormalities with the exception of lubricants and eye washing. A complete evaluation of ocular surface symptoms was performed including the McMonnies Dry Eye Questionnaire, Ocular Surface Disease Index, slit-lamp biomicroscopy, fluorescein break-up time, the fluorescein staining of the cornea and conjunctiva (according to the Oxford grading system), the Schirmer I test, and the meibomian secretion after digital pressure application on the lids. This set of exams was carried out at baseline and repeated at all follow-up assessments. Results: All participants that did not benefit from previous therapies, including corticosteroids tapering schedule, showed an important improvement in the clinical picture after two months with topical cyclosporine medication. Moreover, after 12 months of continuous therapy, all patients showed a clinical improvement in DED signs and symptoms, related to the absence of new RCE episodes. The treatment was well tolerated, and no adverse effects were reported. Conclusion: Although a small number of cases were available of our analysis, the treatment with cyclosporine eye drops represents a promising approach in the management of refractory PEDs with associated ocular comorbidities, since it may reduce the RCE episodes and improve the tear film stability, in absence of systemic or local side effects.