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.

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.

The study evaluated the regenerative responses of the lacrimal functional unit (LFU) after lacrimal gland (LG) ablation. The LG of Wistar rats was submitted to G1) partial LG ablation, G2) partial ablation and transplantation of an allogeneic LG, or G3) total LG ablation, (n = 7-10/group). The eye wipe test, slit lamp image, tear flow, and histology were evaluated. RT-PCR analyzed inflammatory and proliferation mediators. The findings were compared to naïve controls after 1 and 2 months (M1 and M2). G3 presented increased corneal sensitivity, and the 3 groups showed corneal neovascularization. Histology revealed changes in the LG and corneal inflammation. In the LG, there was an increase in MMP-9 mRNA of G1 and G2 at M1 and M2, in RUNX-1 at M1 and M2 in G1, in RUNX-3 mRNA at M1 in G1, and at M2 in G2. TNF-α mRNA rose in the corneas of G1 and G2 at M2. There was an increase in the IL-1β mRNA in the trigeminal ganglion of G1 at M1. Without changes in tear flow or evidence of LG regeneration, LG ablation and grafting are unreliable models for dry eye or LG repair in rats. The surgical manipulation extended inflammation to the LFU.

UNASSIGNED: To assess the long-term (1-year) effect of myopic femtosecond laser-assisted in situ keratomileusis (FSLASIK) on clinical characteristics and tear film biomarkers.
UNASSIGNED: Eighty eyes from 80 patients who underwent FSLASIK were evaluated. Ocular surface symptoms and signs were evaluated using specific questionnaires and tests. The corneal nerves and dendritic cells were examined using in vivo confocal microscopy. Corneal sensitivity was evaluated using a Cochet-Bonnet esthesiometer. Tear inflammatory cytokines and neuropeptides were evaluated using Luminex immunoassay. These examinations were performed preoperatively and at 1, 3, 6, and 12 months postoperatively.
UNASSIGNED: Seventy-three participants completed all follow-up visits. Following FS-LASIK, ocular symptoms and signs (except Schirmer I test) worsened at 1 month but corneal and conjunctival stainings improved by 3 months. The numbers of dendritic cells and activated dendritic cells increased at the 3-month postoperative visit and recovered to preoperative levels by the 6-month visit. Ocular symptoms and corneal sensitivity recovered to preoperative levels at the 12-month visit. Tear break-up time and corneal nerve morphology were not recovered to preoperative status at the 12-month visit. Interleukin (IL)-1β, IL-17A, tumor necrosis factor-α, and substance P tear levels significantly increased at all postoperative visits compared to preoperative levels. Corneal staining scores positively correlated with tear IL-1β and IL-17A levels, whereas corneal nerve morphology positively correlated with corneal sensitivity and negatively correlated with substance P levels.
UNASSIGNED: Although most clinical variables improved at 12 months postoperatively, some tear inflammatory cytokines and substance P remain altered beyond 12 months, indicating that ocular homeostasis is not completely recovered. [J Refract Surg. 2024;40(8):e508-e519.].

UNASSIGNED: The study aimed to investigate the correlation between the change of sex hormone levels and ocular surface parameters in girls with idiopathic central precocious puberty(ICPP).
UNASSIGNED: Eighteen girls with ICPP and 18 age-matched normal girls participated in this study, all of the participants had undergone physical measurements, laboratory tests, imaging examination and ocular surface assessments.
UNASSIGNED: The Objective Scatter Index (OSI) in the ICPP group was significantly higher than in the control group (P = 0.031), girls with ICPP showed slightly lower MNITBUT compared to the normal control group, although this difference was not statistically significant. Bivariate analysis revealed a positive association between estradiol and OSI (r=0.383, P=0.021), Additionally, in the study population, both Luteinizing hormone (LH) and Follicle-stimulating hormone (FSH) were negatively correlated with Mean noninvasive tear breakup time (MNITBUT) (r=-0.359, P=0.031)(r=-0.357, P=0.032).
UNASSIGNED: In comparison with the normal control group, alterations in the OSI were observed in girls with ICPP. This alteration may be associated with an elevation in estrogen levels. Although there was a slight non-significant decrease in NITBUT in ICPP girls, the negative correlation between LH and FSH with MNITBUT suggests new perspective for further investigation.

UNASSIGNED: We determined interblink corneal surface-temperature decline and tear-film evaporation rates of localized tear breakup cold regions (LCRs) and localized tear unbroken warm regions (LWRs) of the corneal surface, as well as that of the overall average corneal surface.
UNASSIGNED: Each subject underwent 4 inter-day visits where the interblink corneal surface-temperature history of the right eye was measured using a FLIR A655sc infrared thermographer. Corneal surface temperature history was analyzed to determine the overall, LCR, and LWR temperature-decline rates. Evaporation rates of LCR and LWR regions were determined from the measured LCR and LWR temperature data using the physical model of Dursch et al.
UNASSIGNED: Twenty subjects completed the study. Mean (SD) difference of LCR temperature-decline rate was -0.08 (0.07)°C/s faster than LWR (P < 0.0001). Similarly, evaporation rates of LCR and LWR were statistically different (P < 0.0001). At ambient temperature, mean LCR and LWR evaporation rates were 76% and 27% of pure water evaporation flux, respectively. There was no statistically significant difference between the inter-day measured temperature-decline rates and the interblink starting temperature.
UNASSIGNED: Significant differences in corneal temperature-decline rate and evaporation rate between LCR and LWR were quantified using infrared thermography. In agreement with literature, LCRs and LWRs correlate directly with fluorescein break-up areas and unbroken tear areas, respectively. Because lipid-evaporation protection is diminished in breakup areas, higher local evaporation rates and faster local cooling rates occur in LCRs relative to LWRs. Our results confirm this phenomenon clinically for the first time.

OBJECTIVE: To determine the diagnostic accuracy of non-invasive tear film break-up time (NIBUT) measured by the handheld lipid layer examination instrument.
METHODS: 108 patients were enrolled in this cross-sectional study and divided into two groups: patients with dry eye (n = 57) categorized by the presence of dry eye symptoms obtained by Schein Questionnaire and minimally-one objective dry eye sign (tear film break-up time <10 s or corneal, conjunctival and lid margin fluorescein staining), and healthy subjects (n = 51).
RESULTS: Dry eye subjects had significantly shorter NIBUT than healthy subjects (6 s vs 20 s, p < 0.001). Logistic regression analysis showed that shorter NIBUT values were excellent indicators of dry eye disease (p < 0.001), with consistency and no significant difference between measurements, even after standardizing the results for age and sex. NIBUT cut-off point to distinguish dry eye from healthy subjects was 12 s (sensitivity 90.2 %, specificity 88.5 %, PPV 92.5 %, NPV 85.2 %, LR +7.82, LR- 0.11, DOR 70.92, DE 89.6 %). Good, but lower accuracy was observed at cut-off value of 10 s (sensitivity 87.8 %, specificity 88.5 %, PPV 92.3 %, NPV 82.1 %, LR+ 7.61, LR- 0.14, DOR 55.2, DE 88.1 %). The area under the ROC curve (AUC) of 0.944 classified NIBUT as a diagnostic test with very high accuracy.
CONCLUSIONS: This study showed a high diagnostic accuracy of NIBUT measured by the handheld lipid layer examination instrument. This simple, reliable, objective and available instrument might regularly take place in routine, standard dry eye diagnostic and can be used by almost every eye specialist.

The impact of viral keratitis (VK) on individuals and society is notable. Early diagnosis and treatment are crucial in managing viral keratitis effectively. Timely intervention with antiviral medications and supportive care can help mitigate the severity of the infection and improve visual outcomes. We examined the prevalence of varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), adenovirus (AdV) and herpes simplex virus type 2 (HSV-2) in patients suspected for ocular infections. Patients included in the study exhibited various clinical manifestations indicative of ocular pathology, such as infectious keratitis, corneal scar, endogenous endophthalmitis, panuveitis, endothelitis, stromal edema, and other relevant conditions. Four different types of tear fluid, corneal samples epithelium, aqueous humor and vitreous humor were taken. After genome extraction, multiplex real-time PCR was used for diagnosis of viruses. 48 (29.6%) out of the total of 162 (100%) eye specimen were positive. The dominant prevalence was VZV (12.3%) and HSV-1 (11.7%) followed by AdV (4.9%) and HSV-2 (0.6%). There were 4 (8.3%) coinfections within the samples (HSV-1 and VZV). Aqueous humor samples demonstrated superior virus detection ability and our only HSV-2 positive sample was from aqueous humor. The utilization of multiplex real-time PCR assays in differential diagnosis of VK holds promise for expeditious diagnoses while also preventing unwarranted antibiotic prescriptions. Moreover, the aqueous humor appears to be a more sensitive site for detecting viral keratitis.

BACKGROUND: Parkinson\'s disease symptoms mostly manifest after significant and irreversible neuropathology. Hence, there is a need to identify biomarkers that can provide indications of disease before significant neuronal degeneration occurs.
OBJECTIVE: To estimate the difference in the concentration of α-synuclein protein in tears between individuals with Parkinson\'s disease and healthy controls.
METHODS: PubMed, Scopus, and Web of Science. The last database search was on December 20, 2023.
METHODS: Primary prospective studies in humans measuring the level of α-synuclein in tears and clinical outcomes reported using mean or median.
METHODS: Individuals with Parkinson\'s disease and healthy controls.
METHODS: The risk of bias was assessed using the Newcastle-Ottawa Scale. The I2 statistic was used to estimate heterogeneity. The outcome measure was the difference in tear total and oligomeric α-synuclein. Mean difference (MD) was used to assess the outcome. The certainty of evidence was rated following the Grading of Recommendations Assessment and Development and Evaluation (GRADE) system.
RESULTS: Three hundred twenty-seven Parkinson\'s disease and 312 healthy control subjects from five studies and 177 Parkinson\'s disease and 166 healthy control subjects from two studies were included in total α-synuclein levels and oligomeric α-synuclein levels analysis, respectively. Total α-synuclein level was not different between Parkinson\'s disease and healthy controls (MD = 0.02 ng/mL [95% confidence interval {CI}: 0.00 to 0.05 ng/mL; I2 = 90%; Z = 1.79; p=0.07; number of studies = 5; GRADE rating = very low]). Stratifying the data based on disease duration, total α-synuclein was higher in subjects with Parkinson\'s disease duration ≥7 years compared with healthy controls (MD = 0.04 ng/mL [95% CI: 0.03 to 0.05 ng/mL; I2 = 0%; Z = 8.24, p<0.00001; number of studies = 2; GRADE rating = low]) but not different between the two groups (MD = -0.12 ng/mL (95% CI: -0.38 to 0.15 ng/mL; I2 = 93%; Z = 0.84, p=0.40; number of studies = 3; GRADE rating = very low]). Oligomeric α-synuclein level was higher in Parkinson\'s disease compared with controls (MD = 6.50 ng/mL [95% CI: 2.79 to 10.20 ng/mL; I2 = 94%; Z = 3.44; p=0.0006; number of studies = 2; GRADE rating = very low]).
CONCLUSIONS: High heterogeneity between studies. Potential sources of heterogeneity could not be explored due to the limited number of studies.
CONCLUSIONS: Tear α-synuclein has the potential to be a noninvasive biomarker for Parkinson\'s disease. Studies are, however, needed to increase certainty in the biomarker and establish how the protein\'s changes in tears correlate with Parkinson\'s disease progression and severity.

Although previous studies have suggested that meteorological factors and air pollutants can cause dry eye disease (DED), few clinical cohort studies have determined the individual and combined effects of these factors on DED. We investigated the effects of meteorological factors (humidity and temperature) and air pollutants [particles with a diameter ≤ 2.5 μ m (PM2.5), ozone (O3), nitrogen dioxide (NO2), and carbon monoxide (CO)] on DED. A retrospective cohort study was conducted on 53 DED patients. DED was evaluated by Symptom Assessment in Dry Eye (SANDE), tear secretion, tear film break-up time (TBUT), ocular staining score (OSS), and tear osmolarity. To explore the individual, non-linear, and joint associations between meteorological factors, air pollutants, and DED parameters, we used generalized linear mixed model (GLMM) and Bayesian kernel machine regression (BKMR). After adjusting for all covariates, lower relative humidity or temperature was associated with a higher SANDE (p < 0.05). Higher PM2.5, O3, and NO2 levels were associated with higher SANDE and tear osmolarity (p < 0.05). Higher O3 levels were associated with lower tear secretion and TBUT, whereas higher NO2 levels were associated with higher OSS (p < 0.05). BKMR analyses indicated that a mixture of meteorological factors and air pollutants was significantly associated with increased SANDE, OSS, tear osmolarity, and decreased tear secretion.