Schirmer Strips Research Articles

A novel Schirmer Strip-Based Tear Matrix Metalloproteinase Measurement in Dry Eye Evaluation.

The diagnosis and evaluation of dry eye require easy-to-use, precise, and consistent tools in clinical setting. Matrix metalloproteinase 9 (MMP-9) has been proven to be a reliable indicator of dry eye inflammation. The aim of this study is to establish an Eu-time resolved fluorescence immunochromatography (Eu-TRFICO) method for quantitative detection of MMP-9 in human tear based upon widely used Schirmer strips. The Eu-TRFICO method for Schirmer strip-based tear MMP-9 measurements were optimized and assembled. The sensitivity, repeatability and homogeneity were evaluated using MMP-9 standard dilutions. The diagnostic and treatment monitoring performance were evaluated in both dry eye patients and normal subjects. The standard curve equation was y = 0.0037+8.0692 / [1+ (x/188.322)-0.8972] (R2 = 0.99998), and the sensitivity was 0.25 ng/mL. The Schirmer strip-based MMP-9 measurements showed acceptable repeatability and homogeneity with different saturation length in both low and high standard solutions. A total of 162 participants (162 eyes) were enrolled in this study, including 41 normal and 121 dry eye subjects. This method exhibited a sensitivity of 74.17% and specificity of 77.5% for dry eye diagnosis, with an AUC value of 0.8275, and cutoff value of 150.67 ng/mL, using normal subjects as negative control. The tear MMP-9 concentrations monitored with this method correlated well with the therapeutic response in dry eye patients. This study developed Eu-TRFICO Schirmer strips with high sensitivity, specificity, precision, and satisfactory clinical testing performance, which provides a convenient and quantitative option for clinical testing of tear MMP-9 in dry eye patients.

Mass spectrometric detection of keratins in tear fluid.

Keratin contamination is a common problem in mass spectrometry proteomic analyses, particularly in bottom-up mass spectrometry. The purpose of this study was to determine the protein contaminants introduced during the proteomic analysis of tear fluid. Human tear fluid samples were collected using Schirmer strips. Proteomic analyses were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on blank Schirmer strips and tear fluid samples, with empty vials serving as controls for assessing environmental contaminant proteins. We detected 26 contaminant proteins (18 keratins and 8 non-keratins). 98.2% of the total protein contamination can be attributed to the 9 keratins, including KRT10 (23.6%), KRT1 (23.5%), KRT2 (15.7%), KRT14 (7.6%), KRT16 (7.0%), KRT5 (6.1%), KRT9 (5.9%), KRT6B (4.6%), and KRT6A (4.3%). A comparison to the proteomic profile of blank Schirmer strips and controls (empty vials) found a strong correlation (R2=0.9753), indicating that these proteins were not from the blank Schirmer strips but are environmental contaminants. On the other hand, several keratins including KRT19, KRT13, KRT4, KRT7, KRT15, KRT8 and KRT18 were present in tear fluid, but either not detected or were negligible in blank strips. Another set of keratins, including KRT5, KRT6A, KRT14, KRT16, and KRT17, were identified as components of tear fluid as well as environmental contaminants. This study revealed nine major contaminant keratins in the mass spectrometry analysis. Several other keratins were identified as constituents of tear fluid. Background subtraction is necessary for the accurate analysis of tear fluid using mass spectrometry.

A simple method (Schirmer IV) to stimulate reflex tear volume secretion by application of gel ice pack on the closed eyelid in dry eye patients

Objectives: We aimed to demonstrate the effectiveness of gel ice pack application on closed eyelids to stimulate reflex tear volume secretion in dry eye patients. Materials and Methods: It is a randomized and controlled study. Sixty patients with dry eyes were randomized into two groups of 30 each: Group A (gel ice pack) and Group B (control group, gel pack at room temperature). Tear secretions using a Schirmer strip were measured before and after the gel pack applications and compared. Results: The study groups (n = 30 in each) were comparable in terms of baseline characteristics such as best-corrected visual acuity (P > 0.05), intraocular pressure (P > 0.05), and baseline tear volume (P > 0.05). In terms of Schirmer’s reading at 5 min, the application of gel ice pack resulted in a significant increase in lacrimation in Group A (25.77 ± 10.76 for right eye [OD] and 26.43 ± 10.67 for left eye [OS]) when compared to Group B (13.43 ± 3.98 for OD and 13.80 ± 4.55 for OS) in the overall patient population (P < 0.001). The pain score (1–5) was slightly higher in the Group A (1, 1, 0–1) when compared to the control group (0, 0, 0–0), which is statistically significant (P < 0.05). No other adverse events were seen in any of the groups. Conclusion: The gel ice pack application on closed eyelids to stimulate reflex tear secretion is a simple, safe, inexpensive, non-invasive, and feasible procedure. Hence, the application of a gel pack (Schirmer IV) stimulate the reflex tear volume secretions could be considered as one of the methods to measure the reflex tear volume secretions.

Development and validation of a novel tool to measure tear volume in volumetric unit (microlitre) per minute "the standard international unit (SI) tear strip".

The Schirmer strip measures tear volume in millimeters. A major limitation of the Schirmer strip is that it does not measure tear volume in a volumetric unit. Furthermore, the linearity range, lower limit of detection (LLOD), and lower limit of quantification (LLOQ) are not established. In this context, we developed a new filter paper based tear volume strip, which measures tear volume in microlitres (a volumetric unit), with a defined linearity range, LLOD, LLOQ, as well as inter-day and intra-day variability. We used standard Whatman filter paper no. 41, measuring 40 × 5 mm. Balanced salt solution (BSS) was used as a tear surrogate. By placing a specific volume of BSS at one marked end of the paper, we measured the distance travelled by the BSS after one minute. Different volumes of BSS (0.5, 1, 2, 4, 8, and 16 μl) were used, and the distance travelled by the BSS drop was recorded. Linearity was evaluated, and the LLOQ and LLOD were determined. Scale data were generated within the linearity range (10 replicates for each). Percentage recovery was calculated, and inter-day and intra-day variation as well as intra examiner and inter examiner coefficients of variation (CV%) were evaluated. High linearity (r2 = 0.976) was observed across all the tested volumes (0.5-16 μl), However, linearity improved within the tested volume range of 1-8 μl (r2 = 0.996). With an LLOD of 0.538 μl and an LLOQ of 1.63 μl, data for the scale were generated from 2-8 μl. Overall, volumetric recovery ranged from 97.34%-104%. The intra-day CV% ranged from 4.576% (for 2 μl) to 1.911% (for 5 μl) and 1.71% (for 7 μl). The inter-day CV% ranged from 1.715 (7 μl) to 4.36% (for 2 μl). The intra examiner CV% ranged from 0.422% (for 7 μl) to 2.66% (for 2 μl). The inter examiner CV% ranged from 1.93% (7 μl) to 3.69% (for 2 μl). This new tear volume measuring tool (named SI tear strip) represents a valuable tool for the measurement of tear volume (in μl) per unit time. This tool may be very helpful for the quantitative evaluation of dry eye patients and functional assessment of lacrimal glands.

Candidate Tear-Based Uveitis Biomarkers in Children with JIA Based on Arthritis Activity and Topical Corticosteroid Use

ABSTRACT Background Uveitis is an inflammatory ocular disease secondary to disruption of the retinal pigmented epithelium (RPE) and blood retinal barrier (BRB). Known clinical factors do not accurately predict uveitis risk in Juvenile Idiopathic Arthritis (JIA). Tear fluid is easily obtained for biomarker study. We aim to identify tear-based markers associated with the presence of uveitis in children with JIA. Methods In a cross-sectional comparative cohort study, tears were collected by Schirmer strips from children with oligoarticular JIA-associated uveitis (JIA-U) and JIA without uveitis (JIA-no-U). A tandem isotope tagging (iTRAQ and TMT) strategy was used for relative quantitation via nanoLC-MS/MS to quantify proteins in the affected eye. Log transformed relative protein abundance of protein levels was compared between groups using Wilcoxon exact test. We explored the influence of arthritis activity and topical corticosteroids (CS) use on protein levels. STRING analysis was performed. Results Tear samples of 14 JIA-U and 14 JIA-no-U patients were analyzed. Thirteen proteins were differentially expressed between both groups. Stratified analysis based on arthritis activity (inactive arthritis) and topical CS (off CS) showed that alpha-2-macroglobulin (p = 0.012), apolipoprotein A1 (p = 0.036), S100A9 (p = 0.05), haptoglobin (p = 0.066), and transthyretin (p = 0.066) consistently differentiated between both groups. On STRING analysis, these proteins were associated with the RPE, BRB, and inflammation. Conclusion Importantly, we identified proteins involved in the RPE, BRB, and immune response that were differentially abundant in the tears of children with JIA-U compared to JIA-no-U, regardless of arthritis activity or topical CS. Candidate tear-based biomarkers may represent a non-invasive means to detect uveitis.

Tear fluid reflects the altered protein expressions of Alzheimer's disease patients in proteins involved in protein repair and clearance system or the regulation of cytoskeleton.

New biomarkers that improve diagnosis of Alzheimer's disease (AD) are warranted. Tear fluid (TF) containing variety of proteins that reflect pathophysiological changes of systemic diseases makes TF proteins potential biomarker candidates for AD. We investigated the expression levels of TF proteins in persons with mild AD and cognitively healthy controls (CO) to find out if altered proteins may link to the AD pathophysiology. We analyzed the data of the 53 study participants (34 COs, mean age 71 and Mini-Mental State Examination (MMSE) 28.9 ± 1.4 and 19 persons with AD, CDR 0.5-1, mean age 71 and MMSE 23.8 ± 2.8). All went through neurological status examination, cognitive tests, and ophthalmological examination. TF was collected using Schirmer strips. The TF protein content was evaluated via mass spectrometry-based proteomics and label-free quantification. Eleven proteins having a role either in protein repair and clearance system, or regulation of cytoskeleton, showed altered expression in AD group compared to CO group. Seven of them were significantly (p ≤ 0.05) upregulated (Sti1, Twf1, Myl6, Otub1, Pls1 and Caza1) or, downregulated (HSP90) in AD group. Altered expression of all these up- or downregulated proteins may be linked to AD pathophysiology. Thus, our results are encouraging for searching new biomarker candidates for AD. TF is potential biomarker candidate, because TF seems to reflect altered protein levels already in mild AD dementia.

Linking human cerebral and ocular waste clearance: Insights from tear fluid and ultra-high field MRI

Impaired cerebral waste clearance (i.e., glymphatics) is evident in aging and neurodegenerative disorders, such as Alzheimer's disease, where an impaired waste clearance system could be related to the accumulation of pathological proteins (e.g., tau). One marker of impaired cerebral clearance is the abundance of enlarged perivascular spaces (PVS). Preclinical studies propose a similar clearance system in the eye, driven by intraocular pressure (IOP). This cross-sectional pilot study explores the link between ocular and cerebral waste clearance by examining the association between MRI-visible PVS, tear fluid total-tau, and IOP.Thirty cognitively healthy participants, all aged over 55 years, underwent 7 Tesla MRI, with PVS visually rated in the centrum semiovale (CSO) and basal ganglia. Tear fluid was collected using paper Schirmer's strips and analyzed for total-tau using enzyme-linked immunosorbent assay. IOP was measured using non-contact tonometry. Partial Spearman's correlation coefficients of eye and brain markers were calculated, adjusted for age, sex, tear fluid-wetting length, and hemispheric region of interest volume.Higher CSO PVS scores in the left and right hemisphere were associated with higher levels of tear fluid total-tau. Higher CSO PVS scores in both hemispheres were related to lower ipsilateral IOP. The exploratory results suggest that higher tear fluid total-tau and a reduced driving force of ocular waste clearance are connected to impaired cerebral waste clearance in cognitive healthy individuals. This study connects the potential ocular glymphatic system to the cerebral waste clearance system. Clarifying waste clearance biology and validating eye biomarkers for cerebral waste clearance could provide treatment targets and diagnostic opportunities for neurological diseases.

A Tear-Based Approach for Rapid Identification of Bacterial Pathogens in Corneal Ulcers Using Nanopore Sequencing.

Corneal ulcers pose a significant threat to vision, with the need for prompt and precise pathogen identification being critical to effective treatment. This study assesses the efficacy of using next-generation portable sequencing (Nanopore Technology) to detect and identify bacterial pathogens directly from tear samples, providing a non-invasive alternative to traditional corneal scraping and culture, which are limited by high false-negative rates. Prospective observational study. Ten participants diagnosed with corneal ulcers. Tear samples were collected from the ocular surface using Schirmer strips. Corneal scrapings and cultures were performed as medically indicated. The 16S rRNA gene was amplified directly from the tear samples using polymerase chain reaction (PCR), and Nanopore sequencing was used for bacterial species identification and taxonomic classification. Comparative analysis was conducted to evaluate the concordance between Nanopore sequencing results and traditional culture methods. Comparison of bacterial species detected via Nanopore sequencing with those identified through traditional culture methods. Bacterial DNA was identified in 8 of the 10 samples analyzed using the tear-based sequencing method. Notably, Nanopore sequencing accurately identified the causative bacteria in all 4 samples that exhibited bacterial growth on culture. Additionally, it detected bacterial pathogens in 2 of the 4 ulcers that did not show bacterial growth on culture. In 2 cases where cultures could not be obtained due to the small size of the ulcer, tear sequencing successfully identified bacterial species, highlighting potentially overlooked pathogens in corneal ulcers. PCR amplification of 16S RNA directly from tears followed by Nanopore sequencing is an effective, non-invasive method to identify bacterial pathogens in corneal ulcers, offering non-inferior results to traditional culture methods. This technique not only allows for the detection of traditionally hard-to-culture organisms, providing immediate diagnostic value to guide treatment, but also enhances our understanding of the microbiological landscape of corneal ulcers, thereby informing more effective treatment strategies.

Ocular tear fluid biomarkers collected by contact lenses

PurposeTo collect tear fluid biomarkers from contact lenses (CLs) and determine the impact of CL wear duration. MethodsRabbits were fitted with commercial etafilcon A CLs, which were collected after 1 min, 4 and 8 h (n = 4/time point). Tear fluid proteins and cytokines were extracted from the CLs and quantified. An exploratory comparison was performed between CLs and Schirmer Strips (SS) for a 1 min duration. ResultsThe concentration of MUC5AC was significantly higher after 4 h of CL wear. The expression of all investigated cytokines (IL-1α, IL-1β, IL-8, IL-17A, IL-21, Leptin, MIP-1β, MMP-9, NCAM-1, and TNF-α) was detectable after 1 min of CL wear, and over time, all showed significant variations throughout the 8-h CL wear period. Notably, IL-1α significantly increased by 8 h of CL wear, while MMP-9 decreased. Albumin and lysozyme did not show significant variations with CL wear. Differences between CLs and SS after 1 min were statistically significant for albumin, Leptin, TNF-α, IL-1α, IL-1β, and IL-8. ConclusionsThe duration of CL wear significantly affects the collection of some tear fluid biomarkers. Albumin, MUC5AC, and cytokines may have individual and synergistic diagnostic or prognostic potential. CLs and SS were similar for lysozyme and MUC5AC but differed in the collection of albumin and some cytokines. CLs are a viable tear fluid collection method for biomarker analyses and can be immediately added as a routine clinical test by being FDA-approved medical devices.

Metabolomic changes in tear fluid following zinc biofortification in the BiZiFED nutritional study: a feasibility study.

Biofortified Zinc Flour to Eliminate Deficiency in Pakistan (BiZiFED) is a nutritional research program that evaluates the impact of consuming zinc biofortified wheat flour on zinc status and associated health outcomes of vulnerable communities in northwest Pakistan. Measuring zinc status from blood samples is fraught with problems. This feasibility study evaluated whether metabolite changes in tear biofluids could be used to understand zinc status. Zinc deficiency is particularly prevalent amongst the female population in Pakistan. Therefore, a crossover trial was developed in which 25 women of reproductive age received standard, wheat flour, and another 25 received zinc-biofortified wheat flour for 8 weeks. At the end of this period, the nutritional intervention was switched between the groups for another 8 weeks. Tear biofluid was collected using Schirmer strips at baseline and after 8 and 16 weeks. Metabolomic analysis was conducted using the MxP® Quant 500 kit on the tear biofluid from a subset of the study participants. Two metabolites had a significantly negative correlation with plasma zinc concentration: tiglylcarnitine and valine. Compared to baseline metabolite concentrations, acetylcarnitine, glutamine, two lysophosphatidylcholines (lysoPC a C16:0 and lysoPC a C18:1), and four sphingomyelins (SM (OH) C16:1, SM C16:0, SM C16:1, and SM C24:0) were all significantly decreased post-zinc intervention, whilst a ceramide (Cer(d18:1/18:0) was significantly increased. These results highlight the potential of using tear biofluids as an alternative source for metabolomic biomarkers, both for the assessment of the zinc status of individuals enrolled in nutritional studies and for indicating physiological changes that arise from nutritional supplementation.

Association Between Dry Eye Disease with Anxiety and Depression Among Medical Sciences Students in Qassim Region: Cortisol Levels in Tears as a Stress Biomarker.

This study aimed to investigate the relationships between anxiety, depression, and ocular surface health. Cortisol levels were detected in human tears, and their relationship with anxiety levels was determined using a validated questionnaire. In total, 112 participants were recruited for this study. All participants were healthy medical students at the Qassim University. Each participant signed an informed consent form after receiving detailed information about the study. Visual acuity examination, TBUT, Shirmer1 test were performed. Participants were asked to fill out three questionnaires: Taylor Manifest Anxiety Scale, Beck Depression Inventory, and The Ocular Surface Disease Index. Tear samples were extracted from the Schirmer strips and cortisol level was measured using ELISA kits. A total of 112 college students were included in the study, 58.9% of whom were females. The mean age was 21.9 ± 1.7 years. Subjective reported symptoms of anxiety levels were significantly correlated with depression scores, the OSDI, and reduced Schirmer test measurements. Moreover, cortisol levels detected in tears were positively associated with higher anxiety scores (r=0.328, P<0.05). Ocular surface health is associated with symptoms of anxiety and depression. The use of tears to measure cortisol levels could be an interesting way to serve as an anxiety biomarker.

Tear cytokines as potential markers of inflammation in feline conjunctivitis.

Conjunctivitis is a prevalent feline ocular surface disorder, often accompanied by inflammation. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), play a crucial role in the pathogenesis of conjunctival inflammation. This study aimed to evaluate the levels of TNF-α and IL-6 in the tears of cats with conjunctivitis and compare them with healthy controls, thereby enhancing our understanding of the inflammatory processes in feline conjunctivitis. Tear samples were collected from cats of various breeds diagnosed with conjunctivitis (n = 15) and healthy control cats (n = 5) using Schirmer strips. The levels of TNF-α and IL-6 were measured using the enzyme-linked immunosorbent assay (ELISA) method. Protein concentration were measured using Bradford assay and data were expressed as pg/mg protein of tear sample. Our results revealed a statistically significant increase in the levels of both TNF-α and IL-6 in cats with conjunctivitis compared to the control group (p < .0001). Positive correlation were observed between tear IL-6 (p < .001, r = 0.902) and TNF-α (p < .001, r = 0.919) with clinical grades of conjunctivitis. The results demonstrated a significant elevation in the levels of TNF-α and IL-6 in the tears of cats with conjunctivitis, suggesting that these cytokines are involved in the inflammatory response of feline conjunctivitis. These findings could pave the way for new diagnostic and therapeutic approaches, focusing on cytokine modulation, to manage feline conjunctivitis more effectively.

Safety and effect of a new artificial saliva containing Zingiber officinale and Cuminum cyminum on stimulating saliva secretion: In vitro, in vivo and preliminary clinical study

IntroductionXerostomia is a symptom that affects quality of life and may lead to malnutrition. While artificial saliva is used as a lubricant for the oral cavity, it rarely stimulates saliva secretion. The objective of this study was to develop a herbal artificial saliva (HAS) that could stimulate saliva secretion and be safe for human consumption. MethodsThree HAS formulations were prepared with 0.3 % Cuminum cyminum extract and 0.05 %, 0.10 %, and 0.15 % Zingiber officinale extract (HAS0.05, HAS 0.10, and HAS0.15, respectively). In vivo skin and oral mucosal irritation tests were used to evaluate the safety of HAS0.15. Additionally, the preliminary efficacy and safety of the three HAS formulations were investigated in 21 healthy volunteers. The sample was sprayed into the oral cavity under the left and right sides of the tongue. Thirty minutes after spraying, the amount of saliva was assessed using a visual analogue scale and a Schirmer strip. In addition, dry mouth symptoms, the saliva pH, and adverse reactions were assessed before and after testing. ResultsHAS0.15 caused minimal skin irritation and did not alter the epithelium or other structures of the oral mucosa. The saliva quantity after using HAS0.10 and HAS0.15 was significantly higher than prior to testing (p < 0.05). None of the formulations induced mucositis or adverse reactions. ConclusionThis study was successful in the development of a new HAS that stimulated saliva secretion and was safe for humans. However, this study is preliminary: Further clinical investigation in patients with hyposalivation will confirm the efficacy of this novel HAS. This novel HAS may be an alternative treatment for the elderly and patients with hyposalivation.

Proteomic Profiling of Tears in Blau Syndrome Patients in Identification of Potential Disease Biomarkers.

Blau syndrome (BS) is a rare autoinflammatory granulomatosis characterized by granulomatous arthritis, uveitis, and dermatitis. Ocular complications are particularly severe in BS, significantly contributing to morbidity. This study aims to identify potential biomarkers for BS ocular degeneration through proteomic profiling of tear samples from affected patients. Seven subjects from the same family, including four carriers of the BS-associated NOD2 mutation (p.E383K), were recruited alongside healthy controls. Tear samples were collected using Schirmer strips and analyzed via mass spectrometry. A total of 387 proteins were identified, with significant differences in protein expression between BS patients, healthy familial subjects, and healthy controls. Key findings include the overexpression of alpha-2-macroglobulin (A2M) and immunoglobulin heavy constant gamma 4 (IGHG4) in BS patients. Bioinformatic analysis revealed that differentially expressed proteins are involved in acute-phase response, extracellular exosome formation, and protein binding. Notably, neutrophils' azurophilic granule components, as azurocidin (AZU1), myeloperoxidases (MPO), and defensins (DEFA3), were highly expressed in the most severely affected subject, suggesting a potential role of neutrophils in BS ocular severity. These proteins might be promising biomarkers for ocular involvement in BS, facilitating early detection and tailored treatment strategies.

Evaluating the Efficacy of the Diluted Schirmer Method for Tear Collection in Dry Eye Syndrome Patients

Purpose This study assesses the diluted Schirmer method’s effectiveness in collecting tears from dry eye syndrome patients, aiming to identify the most suitable tear collection technique for them. Methods A prospective study. Tear samples were collected from patients with dry eye syndrome and healthy individuals using two methods: (1) Direct Schirmer Method: Schirmer strips were directly inserted into the eye to collect tears. (2) Diluted Schirmer Method: After instilling physiological saline into the eye and waiting for 30 s to ensure thorough mixing with tears, Schirmer strips were used for collection. Tear samples from both groups were analyzed and compared for total protein and cytokine levels (IL-1β, IL-6, IL-8, TNF-α). Results (1) The study included 32 participants: 16 with dry eye syndrome (4 males, 12 females, average age 34.92 ± 10.13 years) and 16 healthy controls (5 males, 11 females, average age 32.25 ± 9.87 years). (2) The diluted Schirmer method produced a significantly larger tear volume compared to the direct method (p < 0.05), with lower Visual Analogue Scale (VAS) scores indicating less discomfort (p < 0.05). (3) The average total protein content of the two groups was 51.70 ± 3.166 ng measured by Direct Schirmer method, and the average total protein content of the Diluted Schirmer method was 50.05 ± 3.263 ng. There was no statistical difference between the two groups. (t = 1.051, p = 0.3098) (4) The concentrations of total tear protein and various cytokines measured by both methods were higher in the dry eye group compared to the normal group, with statistically significant differences (p < 0.05). Both methods reflected consistent changes in tear protein profiles. Conclusion The diluted Schirmer method can comfortably collect an adequate volume of tear samples in a short time and consistently reflect changes in tear proteins, making it an effective method for tear collection in patients with dry eye syndrome.

Optimizing Tear Collection in Mice for mRNA and Protein Analysis.

The tear film is a highly dynamic biofluid capable of reflecting pathology-associated molecular changes, not only in the ocular surface but also in other tissues and organs. Molecular analysis of this biofluid offers a non-invasive way to diagnose or monitor diseases, assess medical treatment efficacy, and identify possible biomarkers. Due to the limited sample volume, collecting tear samples requires specific skills and appropriate tools to ensure high quality and maximum efficiency. Various tear sampling methodologies have been described in human studies. In this article, a comprehensive description of an optimized protocol is presented, specifically tailored for extracting tear-related protein information from experimental animal models, especially mice. This method includes the pharmacological stimulation of tear production in 2-month-old mice, followed by sample collection using Schirmer strips and the evaluation of the efficacy and efficiency of the protocol through standard procedures, SDS-PAGE, qPCR, and digital PCR (dPCR). This protocol can be easily adapted for the investigation of the tear protein signature in a variety of experimental paradigms. By establishing an affordable, standardized, and optimized tear sampling protocol for animal models, the aim was to bridge the gap between human and animal research, facilitating translational studies and accelerating advancements in the field of ocular and systemic disease research.

Contact lenses as novel tear fluid sampling vehicles for total RNA isolation, precipitation, and amplification.

The tear fluid is a readily accessible, potential source for biomarkers of disease and could be used to monitor the ocular response to contact lens (CL) wear or ophthalmic pathologies treated by therapeutic CLs. However, the tear fluid remains largely unexplored as a biomarker source for RNA-based molecular analyses. Using a rabbit model, this study sought to determine whether RNA could be collected from commercial CLs and whether the duration of CL wear would impact RNA recovery. The results were referenced to standardized strips of filtered paper (e.g., Shirmer Strips) placed in the inferior fornix. By performing total RNA isolation, precipitation, and amplification with commercial kits and RT-PCR methods, CLs were found to have no significant differences in RNA concentration and purity compared to Schirmer Strips. The study also identified genes that could be used to normalize RNA levels between tear samples. Of the potential control genes or housekeeping genes, GAPDH was the most stable. This study, which to our knowledge has never been done before, provides a methodology for the detection of RNA and gene expression changes from tear fluid that could be used to monitor or study eye diseases.

Discovery and Verification of Sjögren's Syndrome Protein Biomarkers in Tears by Targeted LC-MRM.

Sjögren's syndrome (SS) is an autoimmune rheumatic disorder characterized by exocrine gland dysfunction, mainly from the lacrimal and salivary glands. The disease causes severe aqueous dry eye syndrome (DED) and is associated with high rates of complications, including corneal ulceration, scaring, and perforation. Systemic complications may occur as well as a higher risk of developing lymphoma. Diagnosis of SS-DED is often delayed and difficult to establish. With the aim of discovering biomarkers to help discriminate SS-DED patients, a combination of untargeted and targeted LC-MS/MS analyses were performed on tear samples collected on Schirmer strips and subjected to tryptic digestion. Following the analysis of three cohorts and the development of two targeted LC-sMRM methods for the verification of putative biomarkers found in the first cohort of samples, 64 proteins could be linked to Sjögren's syndrome, in the hopes of helping to confirm diagnoses as well as potentially stratifying the severity of disease in these patients. Proteins that were increased in SS-DED showed activation of the immune system and alterations in homeostasis. Several proteases and protease inhibitors were found to be significantly changing in SS-DED, as well as a consistent decrease in specific proteins known to be secreted by the lacrimal gland.

Schirmer tear test-1 with open or closed eyelids: An evaluation in brachycephalic and nonbrachycephalic dogs.

Assess aqueous tear production when measured with the dogs' eyelids open or closed. Thirty healthy dogs (15 Shih Tzus, 15 Labrador retrievers) were recruited. With the order of testing randomized for each dog, two sessions (separated by 30 min) of STT-1 testing were performed with the dogs' eyelids closed or open. Schirmer strip wetness (every 10 s for 60 s) and number of time(s) the strip dislodged during testing were recorded in each eye. Preferred STT-1 method was surveyed via a global Listserv of the veterinary ophthalmology community. STT-1 values were significantly higher in closed versus open eyes in Shih Tzus (18.6 ± 2.7 mm/min vs. 16.3 ± 2.5 mm/min; p = .002) and Labrador retrievers (21.6 ± 2.9 mm/min vs. 17.8 ± 3.2 mm/min, p < .001), findings that were also significant at times <60 s for either breed (p ≤ .004). Schirmer strips dislodged from six dogs with open eyelids and no dogs with closed eyelids. Maximal STT-1 difference with closed versusopen eyelids was 13 mm/min in Labrador retrievers and 7 mm/min in Shih Tzus. Survey results from 275 veterinarians showed STT-1 performed with "closed eyelids" (38.5%), "open eyelids" (26.9%), or "never paid attention, sometimes closed, sometimes open" (34.6%). Eyelids status (closed or open) during STT-1 testing had a significant impact on aqueous tear secretion in brachycephalic and nonbrachycephalic dogs, highlighting the importance of consistency when repeating STT-1 in a canine patient. STT-1 differences are likely due to sustained reflex tearing throughout the test duration when the dogs' eyelids are closed.

Efficacy ofhydroxypropyl-guar dropsin improving tear film index and ocular surface dynamicsusing two treatment methods under a controlled desiccating environment.

This study aimed to assess the efficacy of hp-guar eye drops on tear film index and ocular surface dynamics under desiccating conditions using protection and relief treatment modalities. The 12 normal, non-dry eye participants were subjected to adverse environmental conditions using a Controlled Environment Chamber (CEC) where the relative humidity (RH) was 5% and the ambient temperature was 21°C. The participants were screened for ocular symptoms, tear osmolarity, ocular surface temperature (OST), tear production using the Ocular Surface Disease Index questionnaire (OSDI), OcuSense TearLab Osmometer, FLIR System ThermaCAM P620, and Schirmer strips. Tear production was calculated by the Tear Function Index test (TFI). The mean tear film osmolarity decreased significantly from 296mOsm/L at 40% RH to 285mOsm/L at 5% RH (p = 0.01). Conflicting responses were seen for osmolarity in protection and relief. Mean tear osmolarity was significantly higher in the protection method in comparison to the relief method (p = 0.005). The mean TFI increased from 557 at 40% to 854 at 5% (p = 0.02). A significant increase in TFI was observed in the relief method in comparison with both 40% (p = 0.001) and 5% (p = 0.04). In the relief method, the mean TFI score went up to 1139 when hp-guar was installed. A significant improvement in ocular comfort was experienced in both the protection (p = 0.041) and relief (p = 0.010) methods at 5% RH. The instillation of hp-guar drops in the relief method resulted in a significant reduction in OST. The mean OST dropped to 33.01 ºC, significantly lower than the recorded OST for both normal (p = 0.040) and dry (p = 0.014) environmental conditions. Hp-guar drops significantly improve tear film parameters under a desiccating environment, however, tear film parameters respond differently to the management modalities. In the protection method, tear film osmolarity was protected against a dry environment, while in the relief mode, an improvement in tear production and a decrease in ocular surface temperature were seen. Hp-guar performance could be maximized for the management of exposure to adverse environments by using a treatment protocol that targets the most affected parameters in each group of patients. Using CEC has the potential to provide researchers with a readily available method to evaluate the efficiency of tear supplementation.