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112 operator platforms9/12/2023 The level or activity of MMP-9 in tear samples has been evaluated frequently and it has been shown that elevated values of this biomarker have a strong and positive correlation and specificity with clinical diagnosis of DED 8, 9, 10. Matrix metallo-proteinases (MMPs) have been shown to alter the corneal epithelium barrier and disease or dysfunction of the lacrimal functional unit alters the balance of MMPs 7. matrix metallo-proteinase 9 (MMP-9), interleukin 17 (IL-17) and intercellular adhesion molecule 1 (ICAM-1). Two major disadvantages of using rats are the low tear volume sample and the lack of existing rat-based multiplex immunoassays for three major biomarkers of interest, i.e. Greater ocular dimension and tear volume made rats superior in this type of research, in comparison to mice. Using rodents like mice and rats has several advantages, such as easier handling, reduced costs, fewer prerequisites for housing and maintenance 6. Even though findings in these animal models cannot literally be extrapolated to humans, it does provide a valuable tool to obtain a first proof-of-concept. To elucidate and to evaluate novel potential treatments for DED, a variety of different animal species were employed to create animal models for DED, including mice, rats, rabbits, dogs, and primates. Research into the biomarker diagnostic field is required to ease the process of fitting the disease into defined parameters 5. Given the complexity of the disease, detailed epidemiological data are lacking, making it very difficult to evaluate the actual prevalence, characteristics, development, and outcomes of this disease 4. Data on aqueous-deficient DED are more difficult to determine. The prevalence of this pathology varies from 3.5 to 68.3% in the population, as reported in several studies 2, 3. DED can be divided into two subtypes: aqueous-deficient and evaporative dry eye characterised by a lack of tear fluid and excessive evaporation of the tear film, respectively 1.Įvaporative DED is characterised by Meibomian gland dysfunction (MGD). It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface. This is the first report of a validated ECLIA that allows measurements of three relevant DED biomarkers in rat tear fluids.ĭry eye disease (DED) is defined as a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. Accuracy and linearity were acceptable for a broader range. Precision met the acceptance criteria in the chosen range: 1062–133 pg/ml for ICAM-1, 275–34.4 pg/ml for IL-17, 1750–219 pg/ml for MMP-9. ECLIA optimum conditions include the use of antibodies at 0.5 µg/ml concentration and 1 h incubation at room temperature with shaking. During optimization, it has observed that incubation time, temperature and agitation affected the robustness of the protocol. Linear mixed, regression models were fit to perform the statistical analysis on the range of concentrations for the chosen analytes. Assays were run based on the U-Plex Meso Scale Diagnostics (MSD) platform, by two independent operators according to the EMA guideline on bioanalytical method validation. Tears, used as matrix, were collected from six healthy Wistar rats. We have developed a multiplex electrochemiluminescence immunoassay (ECLIA) to detect three biomarkers for DED: MMP-9, IL-17 and ICAM-1. Multiplex immunoassays for DED rat research are missing. A major challenge in DED research is detecting multiple biomarkers in a low tear volume sample. Rat models represent valuable tools to study the pathophysiology and to develop novel treatments. Dry eye disease (DED) is a challenge in ophthalmology.
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