Ophthalmic and many systemic diseases may damage the eyes, resulting in changes in the composition and content of biomolecules in ocular biofluids such as aqueous humor and tear. Therefore, the biomolecules in biofluids are potential biomarkers to reveal pathological processes and diagnose diseases. Raman spectroscopy is a non-invasive, label-free, and cost-effective technique to provide chemical bond information of biomolecules and shows great potential in the detection of ocular biofluids. This review demonstrates the applications of Raman spectroscopy technology in detecting biochemical components in aqueous humor and tear, then summarizes the current problems encountered for clinical applications of Raman spectroscopy and looks forward to possible approaches to overcome technical bottlenecks. This work may provide a reference for wider applications of Raman spectroscopy in biofluid detection and inspire new ideas for the diagnosis of diseases using ocular biofluids.

BACKGROUND: Anterior cruciate ligament (ACL) injuries are common in sports and are critical knee injuries that require prompt diagnosis. Magnetic resonance imaging (MRI) is a strong, noninvasive tool for detecting ACL tears, which requires training to read accurately. Clinicians with different experiences in reading MR images require different information for the diagnosis of ACL tears. Artificial intelligence (AI) image processing could be a promising approach in the diagnosis of ACL tears.
OBJECTIVE: This study sought to use AI to (1) diagnose ACL tears from complete MR images, (2) identify torn-ACL images from complete MR images with a diagnosis of ACL tears, and (3) differentiate intact-ACL and torn-ACL MR images from the selected MR images.
METHODS: The sagittal MR images of torn ACL (n=1205) and intact ACL (n=1018) from 800 cases and the complete knee MR images of 200 cases (100 torn ACL and 100 intact ACL) from patients aged 20-40 years were retrospectively collected. An AI approach using a convolutional neural network was applied to build models for the objective. The MR images of 200 independent cases (100 torn ACL and 100 intact ACL) were used as the test set for the models. The MR images of 40 randomly selected cases from the test set were used to compare the reading accuracy of ACL tears between the trained model and clinicians with different levels of experience.
RESULTS: The first model differentiated between torn-ACL, intact-ACL, and other images from complete MR images with an accuracy of 0.9946, and the sensitivity, specificity, precision, and F1-score were 0.9344, 0.9743, 0.8659, and 0.8980, respectively. The final accuracy for ACL-tear diagnosis was 0.96. The model showed a significantly higher reading accuracy than less experienced clinicians. The second model identified torn-ACL images from complete MR images with a diagnosis of ACL tear with an accuracy of 0.9943, and the sensitivity, specificity, precision, and F1-score were 0.9154, 0.9660, 0.8167, and 0.8632, respectively. The third model differentiated torn- and intact-ACL images with an accuracy of 0.9691, and the sensitivity, specificity, precision, and F1-score were 0.9827, 0.9519, 0.9632, and 0.9728, respectively.
CONCLUSIONS: This study demonstrates the feasibility of using an AI approach to provide information to clinicians who need different information from MRI to diagnose ACL tears.

Phthalate plasticizers (PAEs) illegally used in food pose a great threat to human health. A new and efficient sensing platform for the sensitive detection of the PAE residues in biological fluids needs to be designed and developed. Here, we report a simple and reliable surface-enhanced Raman spectroscopy (SERS) active platform with extralong hot spots of Au nanobipyramids@Ag nanorods (Au NBPs@Ag NRs) for the rapid and sensitive detection of PAEs in biological fluids. To achieve high activity, Au NBPs@Ag NRs with different shell lengths were fabricated by controlling the synthesis conditions, and the corresponding SERS properties were investigated by using crystal violet (CryV) and butyl benzyl phthalate (BBP). The experimental results showed that a longer shell length correlated to greater Raman activity, which was confirmed by finite-difference time-domain (FDTD) electromagnetic simulation. More importantly, the extralong hot spots of the Au NBPs@Ag NR SERS-active substrate showed excellent homogeneity and reproducibility for the CryV probe molecules (6.21%), and the detection limit was 10-9 M for both BBP and diethylhexyl phthalate (DEHP). Furthermore, through the standard addition method, an extralong hot spots SERS substrate could achieve highly sensitive detection of BBP and DEHP in serum and tears fluids, and the detection limit was as low as 3.52 × 10-8 M and 2.82 × 10-8 M. Therefore, the Au NBPs@Ag NR substrate with an extraordinarily long surface is efficient and versatile, and can potentially be used for high-efficiency sensing analysis in complex biological fluids.

Thyroid-associated ophthalmopathy (TAO) is the most common orbital disease in adults, with complex clinical manifestations and significant impacts on the life quality of patients. The current diagnosis of TAO lacks reliable biomarkers for early and non-invasive screening and detection, easily leading to poor prognosis. Therefore, it is essential to explore new methods for accurately predicting TAO development in its early stage. In this study, Raman spectroscopy, with non-destructive, label-free, and high-sensitivity characteristics, was used to analyze the differences in biochemical components of orbital adipocyte and tear samples between TAO and control groups. Furthermore, a multivariate analysis method (i.e., Principal Component Analysis-Linear Discriminant Analysis (PCA-LDA)) was applied for data processing and analysis. Compared with controls, PCA-LDA yielded TAO diagnostic accuracies of 72.7% and 75.0% using orbital adipocytes and tears, respectively. Our proof-of-concept results suggest that Raman spectroscopy holds potential for exploring the underlying pathogenesis of TAO, and its potential application in early screening of other thyroid-associated diseases can be further expanded.

Climatic droplet keratopathy (CDK) is a corneal diseases, which is characterized by increased oil-like deposits on the anterior elastic lamina and anterior stromal layer. Severe CDK can even cause blindness, with no specific available treatment. Besides. CDK is poorly understood in terms of its pathogenic mechanisms. Thus, to determine potential biomarkers for CDK, we analyzed the microRNA expression profile in tear samples from CDK patients and investigated their putative roles in the pathogenesis of CDK. Herein, miRNA sequencing and following bioinformatics analysis was performed to explore the roles of their target genes in CDK. A total of 67 differentially expressed miRNAs were identified, of which 25 were upregulated and 42 were downregulated. qPCR verification showed that among the up- and down-regulated miRNAs, expression of five and six, respectively, was most significantly different.The target genes of the differentially expressed miRNAs are involved in the FoxO signaling pathway, tumor necrosis factor (TNF) signaling pathway, and steroid hormone biosynthesis. Protein-protein interaction network analyses identified 20 hub genes, including PTEN, GSK3B, and SMAD3. In conclusion, the panel of differentially expressed miRNAs identified may have potential utility as early diagnostic biomarkers for CDK. Moreover, the TNF signaling pathway is a new potential target in CDK for the development of treatments.

Previous studies have reported that inflammatory cytokine levels increase in the intraocular fluids (aqueous humor and vitreous) of highly myopic eyes, However, there has been currently no study revealing the levels of inflammatory cytokines in tear. Therefore, this study aimed to determine tear cytokine levels of highly myopic eyes, and their relationships with myopic macular degeneration (MMD). This case-control study screened inflammatory cytokines of tear samples from 132 highly myopic and 105 emmetropic eyes using a multiplex cytokine antibody array, and cytokines showing significant intergroup differences were further validated using ProQuantum immunoassays in tear samples from another 60 highly myopic and 60 emmetropic eyes. Ultra-widefield fundus photographs of eyes were classified according to the meta-analyses of the Pathologic Myopia Classification. Associations between tear cytokine levels and MMD category were investigated. As a result, tear levels of interleukin (IL)-6, IL-13 and monocyte chemoattractant protein (MCP)-1 were screened significantly higher in highly myopic eyes than in emmetropic controls (IL-6: 11.70 ± 16.81 versus 8.22 ± 10.76 pg/mL; MCP-1: 63.60 ± 54.40 versus 33.87 ± 43.82 pg/mL; both P < 0.05). Validation assays further demonstrated the elevated concentrations of IL-6 and MCP-1 (IL-6: 13.97 ± 8.41 versus 8.06 ± 7.94 pg/mL, P < 0.001; MCP-1: 32.69 ± 8.41 versus 18.07 ± 8.41 pg/mL, P = 0.003). Tear levels of IL-6 and MCP-1 differed significantly among MMD categories (both P < 0.05). The area under receiver operating characteristic curve were 0.783 and 0.682 respectively (both P < 0.05), when using tear IL-6 and MCP-1 levels to predict the presence of MMD (category ≥2). The ordered logistic regression model also indicated that longer axial length, and higher IL-6 and MCP-1 tear levels were independent predictors of higher MMD category. In our study, highly myopic eyes presented significantly higher levels of tear IL-6 and MCP-1, which may also serve as potential biomarkers for MMD.

OBJECTIVE: To detect proteomic differences in tears between adenoid cystic carcinoma (ACC) and pleomorphic adenoma (PA).
METHODS: Tear samples were collected from 4 patients with ACC, 5 with PA, and 4 control cases. Label-free analysis and parallel reaction monitoring (PRM) were used to screen and validate the tear proteome. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted for bioinformatics analysis.
RESULTS: In total, 1059 proteins in tear samples were identified by label-free analysis. Between ACC and PA, 415 differentially expressed proteins were detected. Based on the GO annotation, enzyme regulator activity and serine-type endopeptidase inhibitor activity in the molecular function category, blood microparticle and extracellular matrix in the cellular component category, and response to nutrient levels in the biological process category were most predominant. By KEGG pathway annotation, the different proteins between ACC and PA mainly participated in complement and coagulation cascades, amoebiasis, African trypanosomiasis and cholesterol metabolism. Eight proteins with mostly significant differences were verified by PRM, and five proteins with more than 10-fold increases in ACC compared with PA, including integrin β, α-2-macroglobulin, epididymal secretory sperm binding protein Li 78p, RAB5C, and complement C5, were identified.
CONCLUSIONS: The combined tools of label-free analysis and PRM are very effective and efficient, especially for samples such as tears. Some proteomic differences in tears between ACC and PA are identified and these protein candidates may be specific biomarkers for future exploration.

OBJECTIVE: The optimal repair method for bursal-side partial-thickness rotator cuff tears (PTRCTs) involving >50% of the thickness remains a controversial topic. The study was aimed to compare the functional and magnetic resonance imaging (MRI) outcomes after in situ repair or tear completion before repair of bursal-side PTRCTs.
METHODS: A retrospective clinical study was conducted involving 58 patients who underwent in situ repair or tear completion before repair of bursal-side PTRCTs between January 2019 and December 2020. These patients were divided into two groups: the in situ repair group and the tear completion before repair group. Functional assessment consisted of active range of motion (ROM), visual analog scale (VAS), American Shoulder and Elbow Surgeons (ASES) score, and Constant-Murley score. The percentages of patients in each group achieving the minimal clinical important difference (MCID) of the functional scores were determined. The healing status of the rotator cuff was assessed by postoperative MRI.
RESULTS: There were no statistically significant differences between the two groups in terms of demographic data. The mean follow-up period was 14.53 ± 2.64 months in the in situ repair group and 15.40 ± 2.66 months in the tear completion before repair group. At the final follow-up, the forward elevation, external rotation, and internal rotation improved significantly in both groups. The VAS, ASES score, and Constant-Murley score improved significantly in the in situ repair group (5.17 ± 2.00 points to 0.11 ± 0.41 points, p = 0.001; 44.04 ± 17.40 points to 95.47 ± 4.32 points, p = 0.001; 49.50 ± 14.38 points to 93.50 ± 3.49 points, p = 0.001) and in the tear completion before repair group (5.43 ± 3.32 points to 0.03 ± 0.18 points, p = 0.001; 41.50 ± 19.59 points to 95.94 ± 2.68 points, p = 0.001; 47.54 ± 17.13 points to 93.97 ± 2.61 points, p = 0.001). Postoperative MRI revealed that the re-tear rate was 7.1% (2/28) in the in situ repair group and 3.3% (1/30) in the tear completion before repair group. No significant differences were observed in terms of the functional scores, the percentages of patients achieving the MCID of the functional scores, and the re-tear rate between the two groups (p > 0.05).
CONCLUSIONS: Both in situ repair and tear completion before repair yielded satisfactory clinical outcomes for patients with bursal-side PTRCTs. No significant differences were observed in the functional and MRI outcomes between the two groups.

Graves\' ophthalmopathy (GO), the most frequent extrathyroidal manifestation of Graves\' disease (GD), can lead to a significant decline in the quality of life in patients. Exosomes, which contain proteins, lipids and DNA, play important roles in the pathological processes of various diseases. However, their roles in Graves\' ophthalmopathy are still unclear. We aimed to isolate exosomes and analyze the different exosomal proteins. Tear fluids were collected from twenty-four GO patients, twenty-four GD patients and sixteen control subjects. The numbers of tear exosomes were assayed using nanoparticle tracking analysis. A Luminex 200 kit and ELISA kit were used to confirm the different cytokine concentrations in serum. Extraocular muscle from GO patients and controls was extracted, and western blotting was used to assay the levels of Caspase-3 and complement C4A. Our study demonstrated that the number of tear exosomes differ from GD patients and control. The expression levels of cytokines, including IL-1 and IL-18, were significantly increased in the tear exosomes and serum from GO patients compared with GD patients and controls. The levels of the exosomal proteins Caspase-3, complement C4A and APOA-IV were significantly increased in GO patients compared to GD patients and controls. Orbital fibroblasts from GO patients showed significantly higher levels of Caspase-3 and complement C4A than those from controls. The levels of serum APOA-IV in GO patients were significantly higher than those in GD patients and controls. Specific proteins showed elevated expression in tear exosomes from GO patients, indicating that they may play important roles in GO pathogenesis.

This study compared the changes in tear inflammatory cytokine levels after intense pulsed light (IPL) combined with meibomian gland expression (MGX) (IPL group) and instant warm compresses combined with MGX (physiotherapy group) as treatments for meibomian gland dysfunction (MGD)-related dry eye disease (DED) to explore their similarities and differences in therapeutic mechanisms.
This study was a post-hoc analysis of a randomized controlled trial. Thirteen patients with MGD-related DED were enrolled in each group and received three treatments correspondingly with 3-week intervals. The levels of 20 tear cytokines, namely, TNF-α, IL-6, MMP-9, CXCL8/IL-8, CXCL10/IP-10, IL-10, EGF, IL-6R, IL-1β, IFN-γ, lactoferrin, Fas ligand, IL-17A, LT-α, S100A9, LCN2/NGAL, IL-13, IL-12/IL-23p40, Fas, and CCL11/Eotaxin, were measured at baseline, before the second and third treatments, and 3 weeks after the third treatment. The primary outcome was the difference in cytokine levels between baseline and the last measurement, and the trends were analyzed at each measurement point.
At the last measurement, a significant decrease was observed in all tear cytokines for both IPL and physiotherapy groups compared with baseline. The IPL group showed greater reductions in IL-6, IL-6R, IL-1β, IL-13, and CCL11/Eotaxin than the physiotherapy group. TNF-α, CXCL8/IL-8, CXCL10/IP-10, IL-10, EGF, IL-1β, IFN-γ, and Lipocalin-2/NGAL levels continued to decrease with treatment time. Important interactions were found in the changes of IL-6 and IL-13 levels, where the levels first decreased and then slightly increased in the physiotherapy group after treatment, while they continued to decrease in the IPL group.
The mechanisms of IPL and physiotherapy in treating MGD-related DED were both associated with reducing inflammation, and the superiority of IPL could be attributed to its better inhibitory effect on inflammatory cytokines like IL-6. In addition, several cytokines were on a downward trend during treatment, suggesting that the vicious cycle of DED was suppressed.