We want to thank all the participants for their help and support in filling out the survey and sending the images of their eyes. The ERC Center grant provided funding for the IRB.

The present study was conducted following the declaration of Helsinki, and the protocol was approved by the Institutional Review Board at Solutions (IRB, 2021/09/14). The study followed the reporting guidelines of the Declaration of Helsinki. All the participants provided informed consent for the questionnaire. The survey was conducted entirely online via the Internet. If the participants met the eligibility requirements, the consent forms, research project flier, and OSDI questionnaire were emailed to them. After submitting the consent forms and completing the OSDI questionnaire, a $10 gift credit card was issued online for completing the survey.
1. OSDI survey for DES evaluation
<ol>
	<li>Use the following criteria: low OSDI score (0-20 points); moderate OSDI score (21-45 points); high OSDI score (46-100points). 
	NOTE: The diagnostic criteria for DES by OSDI score is &#8805;13.</li>
	<li>Ask the eligible subjects to fill out the survey online (Table 1).</li>
</ol>
2. Determination of limbal redness via AOS software
<ol>
	<li>Take a picture of the eyes, one eye at a time. 
	NOTE: The picture has to be clear for the analysis.</li>
	<li>Assign the subject an ID number. Save the subject ID.</li>
	<li>To add a subject, click on the Add tab. 
	NOTE: The system automatically generates the patient ID.</li>
	<li>Fill in the Title tab. Fill in the First name and the surname.</li>
	<li>Fill in the date of birth.</li>
	<li>Fill in the email address, and then confirm the email address.</li>
	<li>Fill in the National Health Service ID (NHS ID) and the Medical Record Number (MRN) number.</li>
	<li>Fill in the Note tab. Then, click on Save.</li>
	<li>Now, select the Subject ID. Then, add/view images. Then, click on + Add.</li>
	<li>Upload the images to the AOS software (see Table of Materials) to evaluate the limbal redness. Upload the right eye first, then the left eye.</li>
	<li>Click on the New Exam tab. Then, click on the Image + tab.</li>
	<li>Add the right eye, and then click on the Selected Media tab.</li>
	<li>Click on the Bulbar icon. Then, click on the Area tab. Click on Analysis Bulbar Redness Grading Scale 0-4.</li>
	<li>Click on the image to start at the limbal area close to the pupil border and follow a pattern to cover the limbal area (Figure 1). Then, click on Save Analysis. 
	NOTE: The redness grade and the % of vessels are saved.</li>
	<li>Click on the Redness Map and Save Analysis.</li>
	<li>Click on Generate Report. Click on both images to generate PDF. 
	NOTE: The PDF report includes the patient&#39;s first name, surname, date of birth, Patient ID, NHS number, and by whom the patient was examined. The PDF report also includes the examination date and examination type.</li>
	<li>Repeat Steps 2.9-2.22; then, add the left-eye image.</li>
	<li>Grade the limbal redness using the AOS software. Analyze the images for bulbar conjunctival redness with a validated objective grading software using the &#34;bulbar redness&#34; function in the limbal areas of the conjunctiva, using an automatic continuous grading from 0-4 in 0.1 unit. 
	NOTE: Grade 0 is minimum redness; Grade 4 is severe redness (Figure 1). Grades 0-1 are coded grade I, Grades 1-2 are coded grade II, Grades 2-3 are coded grade III, and Grades 3-4 are coded grade IV to match the OSDI survey grading system.</li>
</ol>
3. Statistical analysis
<ol>
	<li>Collect the data and analyze the data using Microsoft Excel.
	<ol>
		<li>Summarize the Subject ID, age, gender, race, OSDI score, and the limbal redness in one table.</li>
		<li>Calculate the age, OSDI score, and the limbal redness score mean using Excel software.</li>
	</ol>
	</li>
	<li>Determine the mean &#177; standard deviation of the age, OSDI score, and limbal redness score.</li>
	<li>Calculate the prevalence rate in percentage.
	<ol>
		<li>Divide the number of subjects by the total number of subjects (40), and then multiply by 100.
		<ol>
			<li>Divide the number of subjects with low DES (LDES) by the total number of subjects (40), and then multiply by 100.</li>
			<li>Divide the number of subjects with moderate DES (MODDES) by the total number of subjects (40), and then multiply by 100.</li>
			<li>Divide the number of subjects with severe DES (SDES) by the total number of subjects (40), and then multiply by 100.</li>
		</ol>
		</li>
		<li>Perform the linear regression analysis using Excel software to determine the mean of the two values (x-axis) and the difference between the two means (y-axis). Also, determine the p-value. 
		NOTE: The linear regression analysis determines the relationships between the OSDI score and the AOS redness score.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Meduri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+surface+manifestation+of+COVID-19+and+tear+film+analysis.">Ocular surface manifestation of COVID-19 and tear film analysis.</a> Scientific Reports. 10 (1), 20178 (2020).</li><li>dell'Omo, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+COVID-19-related+lockdown+on+ophthalmic+practice+in+Italy:+A+report+from+39+institutional+centers.">Effect of COVID-19-related lockdown on ophthalmic practice in Italy: A report from 39 institutional centers.</a> European Journal of Ophthalmology. 32 (1), 695-703 (2022).</li><li>Ganne, P., Najeeb, S., Chaitanya, G., Sharma, A., Krishnappa, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+eye+strain+epidemic+amid+COVID-19+pandemic+-+A+cross-sectional+survey.">Digital eye strain epidemic amid COVID-19 pandemic - A cross-sectional survey.</a> Ophthalmic Epidemiology. 28 (4), 285-292 (2021).</li><li>Al-Namaeh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronavirus+disease+pandemic+and+dry+eye+disease:+A+methodology+concern+on+the+causal+relationship.">Coronavirus disease pandemic and dry eye disease: A methodology concern on the causal relationship.</a> Medical Hypothesis Discovery and Innovation in Ophthalmology. 11 (1), 42-43 (2022).</li><li>Hayirci, E., Yagci, A., Palamar, M., Basoglu, O. K., Veral, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+continuous+positive+airway+pressure+treatment+for+obstructive+sleep+apnea+syndrome+on+the+ocular+surface.">The effect of continuous positive airway pressure treatment for obstructive sleep apnea syndrome on the ocular surface.</a> Cornea. 31 (6), 604-608 (2012).</li><li>Giannaccare, G., Vaccaro, S., Mancini, A., Scorcia, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+eye+in+the+COVID-19+era:+how+the+measures+for+controlling+pandemic+might+harm+ocular+surface.">Dry eye in the COVID-19 era: how the measures for controlling pandemic might harm ocular surface.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 258 (11), 2567-2568 (2020).</li><li>Uchino, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+dry+eye+disease+and+its+risk+factors+in+visual+display+terminal+users:+the+Osaka+study.">Prevalence of dry eye disease and its risk factors in visual display terminal users: the Osaka study.</a> American Journal of Ophthalmology. 156 (4), 759-766 (2013).</li><li>Inomata, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+risk+factors+associated+with+diagnosed+and+undiagnosed+symptomatic+dry+eye+using+a+smartphone+application.">Characteristics and risk factors associated with diagnosed and undiagnosed symptomatic dry eye using a smartphone application.</a> JAMA Ophthalmology. 138 (1), 58-68 (2020).</li><li>Schiffman, R. M., Christianson, M. D., Jacobsen, G., Hirsch, J. D., Reis, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+and+validity+of+the+Ocular+Surface+Disease+Index.">Reliability and validity of the Ocular Surface Disease Index.</a> Archives of Ophthalmology. 118 (5), 615-621 (2000).</li><li>Finis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+OSDI+and+SPEED+questionnaires+for+the+evaluation+of+dry+eye+disease+in+clinical+routine.">Comparison of the OSDI and SPEED questionnaires for the evaluation of dry eye disease in clinical routine.</a> Der Ophthalmologe. 111 (11), 1050-1056 (2014).</li><li>Huntjens, B., Basi, M., Nagra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+a+new+objective+grading+software+for+conjunctival+hyperaemia.">Evaluating a new objective grading software for conjunctival hyperaemia.</a> Contact Lens &amp; Anterior Eye. 43 (2), 137-143 (2020).</li><li>Hwang, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy+and+effective+test+to+evaluate+tear-film+stability+for+self-diagnosis+of+dry+eye+syndrome:+blinking+tolerance+time+(BTT).">Easy and effective test to evaluate tear-film stability for self-diagnosis of dry eye syndrome: blinking tolerance time (BTT).</a> BMC Ophthalmology. 20 (1), 438 (2020).</li><li>Wolffsohn, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demographic+and+lifestyle+risk+factors+of+dry+eye+disease+subtypes:+A+cross-sectional+study.">Demographic and lifestyle risk factors of dry eye disease subtypes: A cross-sectional study.</a> The Ocular Surface. 21, 58-63 (2021).</li></ol>

The author has nothing to disclose.

Several previous studies have reported DES using the Schirmer test, tear break-up time (TBUT), and the OSDI score12. The present study used the AOS software to determine the DES using the limbal redness. One of the important critical protocol steps is to have a clear image of the eyes; if the image is blurry, determining the limbal redness is very challenging, and most likely, accurate readings are not obtained. When all the images are collected, one of the troubleshooting techniques is to check t...

COVID-19, caused by a SARS-COV-2 virus infection, was discovered in Wuhan, China, in December 2019. Meduri et al.1 reported a high prevalence rate of mild ocular symptoms in COVID-19 patients. In Italy, eye surgical procedures were reduced due to the pandemic2. Since the outbreak, many have been working from home and wearing masks as a precaution. Each of these elements and the use of digital devices and online learning3 contributed to dry eye syndrome (DES) and eye strain3,4, respectively. Furthermore, there is evidence that wearing masks can cause DES. Wearing the mask may cause tear evaporation and conjunctival discomfort5. Giannaccare et al. reported that 10.3% of the individuals had rising ocular discomfort symptoms during the pandemic, and the mean score of the OSDI was 21, with a mean age of 28.5 years old6.
A cross-sectional study in Japan reported that the percentage of Japanese women who had a combined result of definite or probable dry eye disease was 76.5%, greater than the percentage of men office employees who utilized Visual Display Terminal7. According to Inomata et al., prolonged screen exposure of more than 8 h/day has been linked to the symptomatic dry eye compared to less than 4 h/day8. The OSDI has proven to be a valid and reliable questionnaire for assessing the severity of DES9,10. The AOS software has been used to determine conjunctival hyperemia, and it has been proven to be a very valid software11.
The present study investigated how common DES is in healthy people aged 20-45. An Ocular Surface Disease Index (OSDI) questionnaire was given to 40 people remotely from October 31, 2021, to December 1, 2021, for conducting the test. The AOS and OSDI surveys were used to assess DES. Finally, the two grading methods were compared: the OSDI score and the AOS software. The participants had to first fill up an eligibility questionnaire, which included the following inclusion criteria: (1) Healthy individuals; (2) Age range of 20-45 years; (3) The participants had to be located in the United States.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AOS SOFTWARE</td>
			<td>Advanced Ophthalmic Systems</td>
			<td>SPARCA</td>
			<td>software to access limbal redness</td>
		</tr>
		<tr>
			<td>Microsoft excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>for data collection and analysis</td>
		</tr>
	</tbody>
</table>

comparing objective conjunctival hyperemia grading and the ocular surface disease index score in dry eye syndrome during covid-19

The incidence of dry eye syndrome (DES) has increased due to wearing masks, utilizing digital devices, and working remotely during the pandemic. A survey was conducted during the COVID-19 pandemic to determine the prevalence of dry eye syndrome. A cross-sectional study investigated how prevalent DES is during COVID-19 in healthy patients aged 20-45 in the United States. An Ocular Surface Disease Index (OSDI) questionnaire was given to 40 individuals remotely from October 31, 2021, to December 1, 2021. The AOS and the OSDI survey were used to evaluate DES. The subjects were 29 years old on average (SD 14.14), with 23 males (57.5%) and 17 females (42.5%). According to the OSDI survey, low DES, moderate DES, and severe DES had prevalence rates of 15%, 77.5%, and 7.5%, respectively. White (W) people represent 50% of the population, while African Americans (AA) represent 35%, Asians represent 7.5%, and Hispanics represent 7.5%. Mild DES affected 77.5% of subjects, with 64.50% males and 35.50% females. According to the AOS objective grading system, mild (M) DES, moderate (MO) DES, and severe (S) DES had prevalence rates of 40%, 12.5%, and 15%, respectively. Linear regression was used to compare the two grading systems, and it demonstrated a strong relationship between the two grading systems.

The subjects were 29 years old on average (mean &#177; SD, 29 &#177; 14.14), with 23 males (57.5%) and 17 females (42.5%) (Table 2). White people account for 50% of the population, while African Americans account for 35%, Asians account for 7.5%, and Hispanics account for 7.5% (Figure 2). The average survey score of the OSDI was 6.17 &#177; 6.24, 37.94 &#177; 5.07, 46 &#177; 0 for low, moderate, and high (Figure 3). According to the OSDI Survey,...

Watch this Scientific Journal Video about Comparing Objective Conjunctival Hyperemia Grading and the Ocular Surface Disease Index Score in Dry Eye Syndrome During COVID-19 at JoVE.com

Comparing Objective Conjunctival Hyperemia Grading and the Ocular Surface Disease Index Score in Dry Eye Syndrome During COVID-19

The present protocol describes the cross-sectional research performed on 40 healthy subjects between the ages of 20 and 45 to assess the prevalence of dry eye syndrome (DES) during COVID-19. The OSDI survey evaluated DES, and the advanced ophthalmic systems (AOS) software was used to assess limbal redness.

The incidence of dry eye syndrome (DES) has increased due to wearing masks, utilizing digital devices, and working remotely during the pandemic. A survey ...

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2.2K Views.  Eye Research Center, Wilmington, DE. The present protocol describes the cross-sectional research performed on 40 healthy subjects between the ages of 20 and 45 to assess the prevalence of dry eye syndrome (DES) during COVID-19. The OSDI survey evaluated DES, and the advanced ophthalmic systems (AOS) software was used to assess limbal redness.

Video: Comparing Objective Conjunctival Hyperemia Grading and the Ocular Surface Disease Index Score in Dry Eye Syndrome During COVID-19

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...

Inverse Probability of Treatment Weighting (Propensity Score) using the Military Health System Data Repository and National Death Index

When randomized controlled trials are not feasible, retrospective studies using big data provide an efficient and cost-effective alternative, though they are at risk for treatment selection bias. Treatment selection bias occurs in a non-randomized study when treatment selection is based on pre-treatment characteristics that are also associated with the outcome. These pre-treatment characteristics, or confounders, can influence evaluation of a treatment&#39;s effect on the outcome. Propensity scores minimize this bias by balancing the known confounders between treatment groups. There are a few approaches to performing propensity score analyses, including stratifying by the propensity score, propensity matching, and inverse probability of treatment weighting (IPTW). Described here is the use of IPTW to balance baseline comorbidities in a cohort of patients within the US Military Health System Data Repository (MDR). The MDR is a relatively optimal data source, as it provides a contained cohort in which nearly complete information on inpatient and outpatient services is available for eligible beneficiaries. Outlined below is the use of the MDR supplemented with information from the national death index to provide robust mortality data. Also provided are suggestions for using administrative data. Finally, the protocol shares an SAS code for using IPTW to balance known confounders and plot the cumulative incidence function for the outcome of interest.

Inverse Probability of Treatment Weighting Propensity Score using the Military Health System Data Repository and National Death Index

When randomized controlled trials are not feasible, a comprehensive health care data source like the Military Health System Data Repository provides an attractive alternative for retrospective analyses. Incorporating mortality data from the national death index and balancing differences between groups using propensity weighting helps reduce biases inherent in retrospective designs.

When randomized controlled trials are not feasible, retrospective studies using big data provide an efficient and cost-effective alternative, though they ...

Customizing a Cryolite Glass Prosthetic Eye

In Germany, Austria, and Switzerland, over 90% of ocularists still manufacture customized prostheses using cryolite glass from Thuringia. The present manuscript demonstrates this long-forgotten technique in detail. This manuscript shows some major advantages of manufacturing prosthetic eyes using cryolite glass in comparison to poly(methyl methacrylate) (PMMA). These advantages include a lighter weight of the prosthesis, higher levels of patient satisfaction, and only one appointment necessary for the customized manufacturing. Potential risk of breakage seems not to be a critical disadvantage for glass prosthetic eye wearers. However, in some patients, manufacturing a well-fitting prosthetic eye is not possible or reasonable due to anophthalmic socket complications such as post nucleation socket syndrome, scarred fornices, or an orbital implant exposure. This article gives ophthalmologists a better insight into ocularistic care in order to improve the essential interprofessional collaboration between ocularists and ophthalmologists.

This manuscript shows each step of customizing a cryolite glass prosthetic eye including some major advantages of the use of cryolite glass for manufacturing an eye prosthesis compared to poly(methyl methacrylate). In addition, this manuscript gives ophthalmologists better insight into the ocularistic care that could improve interprofessional collaboration.

In Germany, Austria, and Switzerland, over 90% of ocularists still manufacture customized prostheses using cryolite glass from Thuringia. The present ...

Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing the difference in corneal reflection generated by the phone camera flash relative to the iris center based on high resolution images, the app can measure misalignment with a much higher precision than the naked eye performing the Hirschberg test. It has been validated in a previous clinical evaluation study by comparing to the clinical gold standard-prism and alternate cover test. The goal of this article is to describe the testing techniques regarding how to use the app to measure ocular alignment for different fixation distances, without or with cover to break fusion, as well as angle kappa, so that users can use the app to perform equivalent tests typically done in clinic using prisms.

We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 &#181;g/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) &#8804; 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

This article describes a convenient method for monitoring dynamic changes in antibody titers for two immunoglobulin isotypes concurrently (either IgA, IgM, or IgG) resulting from an immune response to SARS-CoV-2 infection or vaccination. This 'Multianalyte Covid-19 Immune Response Panel' employs three indirect immunoassays built on coded microspheres that are read using a flow-based multiplex reader with 'dual-channel' capability.

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes ...

Bridging the Technology Divide in the COVID-19 Era: Using Virtual Outreach to Expose Middle and High School Students to Imaging Technology

Increasing the diversity of students choosing careers in science, technology, engineering, and mathematics (STEM) fields is an area of intense focus across the USA, especially in kindergarten through 12th grade (K-12)-focused pipeline programs in medical schools. A diverse STEM workforce contributes to better problem-solving and equity in health care. Two of the many major barriers for rural students are the lack of sufficient STEM role models and limited access to technology in the classroom. Medical schools often serve as an important resource for students in the local community who can easily gain access to STEM professionals and modern technology through on-campus, sponsored events and STEM outreach to the local classrooms. However, underrepresented minority (URM) students often live in socioeconomically distressed parts of rural states such as Arkansas, where access to STEM role models and technology is limited. Virtual learning in the COVID-19 era has proven that the imaging technology resources of a medical school can be harnessed to reach a wider audience, especially students living in rural areas far from the medical school campus.

This article presents an overview of how synchronous web-based virtual outreach can be used to expose 6th-12th grade students to advanced imaging technologies such as ultrasound, computerized tomography, and electroencephalography. The paper discusses the methods and equipment needed to livestream integrated educational sessions for effective student engagement in STEM.

Increasing the diversity of students choosing careers in science, technology, engineering, and mathematics (STEM) fields is an area of intense focus ...

Advancing Cryostorage of Human Stem Cell-Derived Retinal Pigment Epithelial Cells

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of&#160;RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2&#8242;-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

Author Spotlight: Development of an Efficient and Feasible Method of Retinal Pigment Epithelial Cell Freezing

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in ...

Scopolamine-Induced Lacrimal Gland Dysfunction in Rats

A Rat Dry Eye Model with Lacrimal Gland Dysfunction Induced by Scopolamine

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal tear production can lead to a disturbance in the ocular surface environment, including corneal damage and inflammation. In severe cases, ADDE can cause vision loss or even blindness. Currently, dry eye treatment is limited to eye drops or physical therapy, which can only alleviate eye discomfort symptoms and cannot fundamentally cure dry eye syndrome. To restore the function of the lacrimal gland in dry eye, we have created an animal model of lacrimal gland dysfunction in rats induced by scopolamine. Through the comprehensive evaluation of the lacrimal gland, corneas, conjunctivas, and other factors, we aim to provide a full understanding of the pathological changes of ADDE. Compared with the current dry eye mouse model, this ADDE animal model includes a functional evaluation of the lacrimal gland, providing a better platform for studying lacrimal gland dysfunction in ADDE.

Author Spotlight: Challenges in Developing Dry Eye Animal Models and Future Research Directions

Here, we establish a rat model of lacrimal gland dysfunction to provide a basis for the study of aqueous-deficient dry eye.

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal ...