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 the images for clarity. One of the software limitations is that if the image is blurry, the readings would not be accurate, and this subject could not be used in this case.
The current study determines the relationship between the OSDI score and the AOS limbal redness. The significance of using the AOS limbal redness is that it adds more information to the OSDI score, such as the scale of the limbal redness in relation to the OSDI score.
In our experience, using both scores, the OSDI survey, and the AOS software, is critical for a successful prevalence rate of DES. The AOS limbal redness survey determines the AOS limbal redness, and the OSDI survey determines the DES grade, which can be low, moderate, or severe. In the present study, a 0-1 redness scale for grade 0, a 1-2 redness scale for grade 1, a 2-3 redness scale for grade 2, and a 3-4 redness scale for grade 3 were used. Grade 0 indicates healthy eyes with no redness, while grade 3 indicates severe redness with severe DES. The use of digital screens has been shown to decrease TBUT, ocular surface staining, and signs of meibomian gland dysfunction, all of which contribute to DES13.
In summary, the prevalence rate of 40 healthy subjects between the ages of 20-45 is high. To assess the prevalence rate of DES during COVID-19, the OSDI survey was used to evaluate DES, and AOS software was used to evaluate limbal redness. In addition, the relationship between the OSDI and AOS is linear. Finally, the AOS software could be used to determine DES and add more information to the OSDI score and TBUT. However, to determine the prevalence rate of DES in a large cohort, future studies need to be performed, including the OSDI score, TBUT score, and the AOS software. In addition, it is still necessary to further verify the reliability and applicability of the AOS software concerning the OSDI score and TBUT score before using both models in day-to-day clinical practice.

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><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, low DES, moderate DES, and severe DES had prevalence rates of 15%, 77.5%, and 7.5%, respectively (Figure 4). Mild DES affects 77.5% of subjects, with males accounting for 64.50% and females accounting for 35.50%. The average redness score of the AOS was 0.47 &#177; 0.23, 1.50 &#177; 0.28, 2.60 &#177; 0.40, 3.65 &#177; 0.28, for Grade 0, Grade 1, Grade 2, and Grade 3 (Figure 5). According to the AOS objective grading system, mild DES, moderate DES, and severe DES had prevalence rates of 27.5%, 12.5%, and 10%, respectively (Figure 6). Linear regression was used to compare the two grading systems, and it demonstrated a strong relationship between them, with P &#60; 0.001 statistically significant (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63812/63812fig01.jpg" /> 
Figure 1: An example of the bulbar redness scale examination type. (A) Illustrates the redness scale. (B) Illustrates the vessels percentage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63812/63812fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63812/63812fig02.jpg" /> 
Figure 2: Pie chart showing the ethnicity of 40 healthy subjects. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63812/63812fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63812/63812fig03.jpg" /> 
Figure 3: The mean of the OSDI score of the low, moderate, and high DES. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63812/63812fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63812/63812fig04.jpg" /> 
Figure 4: Pie chart showing the percentage of participants&#39; OSDI scores. The score reflects low dry eye syndrome (LDES), moderate dry eye syndrome (MODDES) score, and a severe dry eye syndrome (SDES) score. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63812/63812fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63812/63812fig05.jpg" /> 
Figure 5: The mean of the AOS redness grading scale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63812/63812fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63812/63812fig06.jpg" /> 
Figure 6: The AOS software&#39;s redness score. The score shows the percentage of participants with a healthy score (H), a mild dry eye syndrome (MDES) score, a moderate dry eye syndrome (MODDES) score, and a severe dry eye syndrome (SDES) score. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63812/63812fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63812/63812fig07.jpg" /> 
Figure 7: Scatter plot and linear regression comparing the OSDI score to the AOS software redness grading score. P &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63812/63812fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Ocular Surface Disease Index (OSDI) questionnaire used in the study. Low OSDI score (0-20 points); Moderate OSDI score (21-45 points); High OSDI score ( 46-100 points). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63812/63812_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Participants&#39; demographics and the grading score. One subject was 18 years old. White (W), African Americans (AA), Asians (A), Hispanics(H). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63812/63812_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

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 ...

comparing-objective-conjunctival-hyperemia-grading-ocular-surface

Nanyang Technological University

Research

JoVE Journal

Medicine

2.3K Views.  Eye Research Center, Wilmington, DE. Momenteel, hoewel OSDI-onderzoek vaak wordt gebruikt om het droge ogensyndroom te beoordelen, zal de AOS-software worden gebruikt om het droge ogensyndroom objectief te bepalen met behulp van het roodheidsclassificatiesysteem. Het belangrijkste voordeel van het gebruik van de AOS-software is om het droge ogen syndroom objectief te beoordelen. Voor het onderzoeken van de prevalentie van droge ogen syndroom, of DES, e-mail de Ocular Surface Disease Index, of OSDI-vragenlijst, naar de deelnemers...

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

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

Immunology and Infection

Intense Pulsed Light for the Treatment of Dry Eye Owing to Meibomian Gland Dysfunction

Dry eye disease (DED) is an increasingly common condition and one of the most common complaints of patients. The vast majority of DED is caused by the so-called &#34;evaporative&#34; subtype, that is mainly caused by meibomian gland dysfunction (MGD). Intense pulsed light (IPL) devices employ high intensity pulses of polychromatic lights with a broad range of wavelength (515-1200 nm). IPL treatment has been utilized for years in the field of dermatology, and then its use was applied to ophthalmology for the treatment of MGD. Recently, a new device employing IPL was specifically designed for the periocular application. This procedure determines the thermal selective coagulation and ablation of superficial blood vessels and telangiectasias of the eyelids skin, reducing the release of inflammatory mediators and tear cytokines levels, and improving meibomian glands outflow. IPL treatment is noninvasive and easy to perform, lasts for only a few minutes and can be conducted in an office setting. In the present study, 19 patients underwent 3 sessions of IPL treatment. After treatment, both mean noninvasive break-up time and lipid layer thickness grade significantly increased, as a result of an improvement of tear film stability and quality, respectively. Conversely, no statistically significant changes were found for meibomian gland loss and tear osmolarity. Furthermore, the vast majority of the treated patients (17/19; 89.5% of the total) perceived an improvement of their ocular discomfort symptoms after IPL treatment. Although IPL treatment provides an improvement of both ocular surface parameters and ocular discomfort symptoms after one cycle of three sessions, regular repeated treatments are usually required to maintain the persistence over the time of its beneficial effects.

Dry eye disease is an increasingly common condition, which strongly impair patients&#39; life quality. Recently, a new device employing intense pulsed light, specifically designed for the periocular area, has been shown to improve tear film stability and ocular discomfort symptoms in dry eye disease owing to meibomian gland dysfunction.

Dry eye disease (DED) is an increasingly common condition and one of the most common complaints of patients. The vast majority of DED is caused by the ...

Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &ndash; Adaptation for the COVID-19 Pandemic

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed due to challenges with different diagnostic modalities. Ciliary videomicroscopy, using Digital High-Speed Videomicroscopy (DHSV), one of the diagnostic tools for PCD, is considered the optimal method to perform ciliary functional analysis (CFA), comprising of ciliary beat frequency (CBF) and beat pattern (CBP) analysis. However, DHSV lacks standardized, published operating procedure for processing and analyzing samples. It also uses living respiratory epithelium, a significant infection control issue during the COVID-19 pandemic. To continue providing a diagnostic service during this health crisis, the ciliary videomicroscopy protocol has been adapted to include adequate infection control measures.
Here, we describe a revised protocol for sampling and laboratory processing of ciliated respiratory samples, highlighting adaptations made to comply with COVID-19 infection control measures. Representative results of CFA from nasal brushing samples obtained from 16 healthy subjects, processed and analyzed according to this protocol, are described. We also illustrate the importance of obtaining and processing optimal quality epithelial ciliated strips, as samples not meeting quality selection criteria do now allow for CFA, potentially decreasing the diagnostic reliability and the efficiency of this technique.

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &#8211; Adaptation for the COVID-19 Pandemic

To guarantee a successful and high-quality ciliary functional analysis for PCD diagnosis, a precise and careful method for respiratory epithelium sampling and processing is essential. To continue providing PCD diagnostic service during the COVID-19 pandemic, the ciliary videomicroscopy protocol has been updated to include appropriate infection control measures.

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed ...

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and faster. It creates low spatial frequency/high temporal frequency bright stimuli and records cortical activity initiated primarily by afferents in the magnocellular ON pathway. This pathway contains neurons with larger volumes and axonal diameters, and it is preferentially damaged in early glaucoma, which can result in visual field loss. The study presented here uses standard operative procedures (SOP) of icVEP to obtain reliable results. It can detect visual function loss using a signal-to-noise ratio (SNR) corresponding to the defects of retinal nerve fiber layer (RNFL) in early stage open-angle glaucoma (OAG). A setting of 10 Hz and condition of 15% positive-contrast (bright) are selected to differentiate OAG patients and control subjects, with each check containing eight runs. Each run persists for 2 s (for 20 total cycles). A flowchart is constructed, which consists of pupil size and intraocular pressure over a 30 min rest period before each examination. Additionally, the testing order of eyes is performed to obtain reliable electroencephalographic signals. VEPs are recorded and analyzed automatically by software, and SNRs are derived based on a multivariate statistic. An SNR of &#8804; 1 is considered abnormal. A receiver-operating-characteristic (ROC) curve is applied to analyze the accuracy of group classification. Then, the SOP is applied in a cross-sectional study, showing that icVEP can detect glaucomatous visual function abnormality in the central visual field in the form of SNR. This value also correlates with the thickness thinning of RNFL and produces high classification accuracy for early stage OAG. Thus, it serves as a useful and objective diagnostic technology for the early detection of glaucoma.

The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and ...

Neuroscience

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

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 ...

Assessing the Health of the Corneal Nerves in Ocular Surface Diseases

Corneal Sensitivity Testing Procedure for Ophthalmologic and Optometric Patients

The cornea is the most densely innervated structure in the human body, making it one of the most sensitive tissues. Changes in corneal nerve sensitivity can be observed in several ocular surface diseases. Nerve sensitivity may be increased, as is often seen in patients with a neuropathic component to ocular pain, or decreased, as is seen in patients with neurotrophic keratitis. Corneal sensitivity testing involves assessing a patient's reaction to brief corneal stimulation, yields insight into the health of the corneal nerves, and provides diagnostic value for evaluating the health of the nerves and the interplay with the ocular surface. Currently, there is limited published guidance on how to conduct corneal sensitivity testing in a clinical setting. This article presents a protocol for testing corneal sensitivity using easy-to-use, low-cost materials that are readily accessible to eye care providers (either a cotton swab, a piece of dental floss, or a finely tapered tissue). This protocol allows for qualitative assessment of corneal sensitivity in which responses to corneal stimulation are rated from 0 (no response) to 3 (hypersensitive response). This test can be performed quickly (in approximately 30 s). Given its diagnostic value and accessibility, corneal sensitivity testing should be included as part of the standard eye examination for any patient undergoing an ocular surface examination.

 Author Spotlight: Establishing a Practical and Cost-Effective Protocol for Corneal Sensitivity Testing in Clinical Settings

Corneal sensitivity testing provides insight into the health of the corneal nerves and helps diagnose ocular surface diseases. We present a concise protocol to qualitatively assess corneal sensitivity that can be readily used by eye care providers across clinical settings.

The cornea is the most densely innervated structure in the human body, making it one of the most sensitive tissues. Changes in corneal nerve sensitivity ...

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its underlying mechanisms. Therefore, we developed a mouse model of dry eye disease by surgically removing both the extraorbital lacrimal glands (ELG) and intraorbital lacrimal glands (ILG) to investigate the ocular surface pathology in the context of aqueous deficiency dry eye. Two weeks post operation, the mice exhibited severe dry eye manifestations, including reduced tear secretion, corneal epithelial irregularities, positive fluorescein sodium staining, and neovascularization. Histological examination via hematoxylin and eosin staining revealed inflammatory cell infiltration and corneal epithelium dysplasia. Immunofluorescence staining and quantitative reverse-transcription polymerase chain reaction revealed decreased expression of the normal corneal epithelial biomarkers K12 and Pax6 and increased expression of Sprr1b in the corneal epithelium. These ocular manifestations indicated abnormal corneal epithelial differentiation. Furthermore, immunofluorescence staining of Ki67 revealed the increasing cell proliferation. In conclusion, the ELG plus ILG excision model proved suitable for studying changes in the ocular surface and elucidating the mechanisms underlying aqueous deficiency dry eye.

Here, we present a method to establish a mouse model of aqueous-deficient dry eye by excising the extraorbital and infraorbital lacrimal glands and evaluate the changes in the ocular surface in aqueous deficiency dry eye.

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its ...

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.

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

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