The role of MMPS and TIMPS in the feline ocular surface system during corneal epithelial wound healing

The ocular surface system is a complex microenvironment including the cornea, conjunctiva, lacrimal gland, meibomian glands and tear film. Matrix metalloproteinases (MMPs), a family of zinc- and calcium-dependent proteinases involved in maintaining and remodelling extracellular matrix and inflammation, have been found in ocular tissues and tears. The analysis of tears has been used to identify potential biomarkers for eye diseases. Contributions of individual ocular surface components to tear proteins are not fully understood. This study investigated the expression of MMPs and tissue inhibitors of MMPs (TIMPs) (particularly MMP-9 and -2) in tears and ocular tissues following corneal epithelial wounding (debridement) at predetermined time points using a feline model. Preliminary work validated the use of eye-flush tears (collected after instillation of saline) and procedural control eyes (same treatment as wounded eyes) as controls for the tear collection study. Gelatin zymography and immunohistochemistry were used to detect MMPs and TIMPs in tears and ocular surface tissues, and data was then analysed using statistical methods. During corneal healing, expression of MMP-9 was detected in all ocular surface components with significant increase in tears, the corneal and limbal epithelium, keratocytes and conjunctival epithelium. In contrast, MMP-2 was found significantly increased only in tears and the corneal and conjunctival epithelium. Analysis of tears from wounded and procedural control eyes suggested a minimal contribution of the healing corneal epithelium to tear-derived MMP-9 and -2. Other possible contributors to increased tear MMP-9 levels included the lacrimal gland, inflammatory cells in conjunctival tissue including conjunctival-associated lymphoid tissue (CALT). Expression of MMP-1, TIMP-1 and -2 in ocular tissues in response to wounding were also investigated. MMP-1 was increased in the healing corneal epithelium, keratocytes and limbal epithelium. TIMP-1 and -2 immunoreactivity paralleled that of MMP-9 in the cornea and lacrimal gland. The current study revealed the complexity of interactions in the corneal epithelial wound healing processes, as exemplified for MMP-9 and -2 with simultaneous increases in tears and ocular surface tissues in response to corneal wounding, and subsequent regulation during wound healing. These observations highlight the importance of considering the ocular surface microenvironment in its entirety for future studies

A preliminary study of changes in tear film proteins in the feline eye following nictitating membrane removal

Objective To investigate the influence of nictitating membrane (third eyelid) removal on selected proteins in feline tears. Animal studied Domestic short-haired cats (7-17months; 2.6-5.2kg) were used. Procedures Eye-flush tears were collected periodically for up to 18weeks from both eyes of animals with nictitating membranes removed, but nictitating gland left intact, (n=4) or with nictitating membranes intact (n=4). Tear comparisons were based on total protein content (TPC) using micro bicinchoninic acid assay, immunoglobulin A (IgA), and matrix-metalloproteinase (MMP)-9 measurements using sandwich enzyme-linked immunosorbent assay (ELISA) and tear gelatinase activity using gelatin zymography. Expression of MMP-2 and -9 in nictitating membranes removed at baseline (week 0) and eyes collected at 18weeks were also investigated in histological sections using immunoperoxidase for visualization. Results Nictitating membrane removal did not significantly change TPC and MMP-9 in tears within the first 4weeks. MMP-9 was not detected by ELISA in tears from eyes without nictitating membranes from week 5 onwards. IgA (%IgA of TPC) data varied between animals. Gelatin zymography showed increased MMP-2 and -9 activity in tears from eyes without nictitating membranes at week1 and a decrease following week 2 post-surgery. MMP-2 and -9 were immunolocalised to conjunctival goblet cells of removed nictitating membranes and to the conjunctival epithelium, respectively. After 18weeks, the distribution of MMPs in tissue was comparable between eyes with and without nictitating membranes. Conclusions Based on this preliminary study, nictitating membrane removal appeared to cause long-term changes in expression of tear proteins, including reduced MMP-9 expression

A comparison of basal and eye-flush tears for the analysis of cat tear proteins

Purpose: To identify a rapid and effective tear collection method providing sufficient tear volume and total protein content (TPC) for analysis of individual proteins in cats. Methods: Domestic adult short-haired cats (12-37 months; 2.7-6.6 kg) were used in the study. Basal tears without stimulation and eye-flush tears after instillation of saline (10 ÃŽ l) were collected using microcapillary tubes from animal eyes either unwounded control or wounded with 9-mm central epithelial debridement giving four groups with n = 3. Tear comparisons were based on total time and rate for tear collection, TPC using micro bicinchoninic acid (BCA), tear immunoglobulin A (IgA), total matrix-metalloproteinase (MMP)-9 concentration using sandwich enzyme-linked immunosorbent assay (ELISA) and MMP-9 activity. Results: Eye-flush tears were collected significantly faster than basal tears in wounded eyes with higher rates for tear collection in unwounded control and wounded eyes. TPC was significantly lower in eye-flush tears compared to basal tears. The relative proportion of tear IgA normalized to TPC (% IgA of TPC) was not significantly different between basal and eye-flush tears. In unwounded control eyes, MMP-9 was slightly higher in eye-flush than in basal tears; activity of MMP-9 in both tear types was similar. In wounded eyes, eye-flush tears showed highest MMP-9 levels and activity on Day 1, which subsequently decreased to Day 7. MMP-9 activity in basal tears from wounded eyes did not display changes in expression. Conclusions: Eye-flush tears can be collected rapidly providing sufficient tear volume and TPC. This study also indicates that eye-flush tears may be more suitable than basal tears for the analysis of MMPs following corneal wounding

Assessment and Management of Dry Eye Patients for Non-Ophthalmic Healthcare Practitioners

Dry eye is a very common condition in many parts of the world. Both ophthalmologists and other healthcare professionals, such as optometrists and general practitioners, are often involved in the care of these patients. The main thrust of dry eye management is over-the-counter tear lubricants. There is a wide range of tear lubricants available today. These lubricants vary in viscosity, duration of action, type of preservatives, osmolarity/osmolality and pH. This article aims to educate healthcare professionals on 1) how to assess and manage patients with mild to moderate dry eyes and 2) how to identify patients who may need a referral to an ophthalmologist. Strategies on the use of available treatments and their limitations, as well as factors that may affect patient compliance will be discussed

MMP-2 and MMP-9 activity in tears.

<p>Activity of proMMP-2, proMMP-9 and MMP-9 in tears from wounded and procedural control eyes at different phases of epithelial healing.</p>1<p>Optical Density (OD) multiplied by the number of gel pixels.</p>*<p>significantly greater MMP activity when compared to before wounding/procedure (p<0.001).</p>†<p>significantly greater proMMP-9 activity in wounded eyes as compared to procedural control eyes (p<0.04).</p

MMP-2 expression in ocular surface tissues.

<p>Representative light micrographs of tissue sections showing MMP-2 expression (A–D) corneal epithelium, (E–H) stromal keratocytes, (I–L) bulbar conjunctival epithelium, (M–P) lacrimal gland and (Q–T) meibomian gland of unwounded control eyes collected prior to surgery and wounded eyes at various phases of healing. Arrows indicate stromal keratocytes. Arrow head shows an example of a conjunctival goblet cell.</p

Application of intrinsic time-scale decomposition (ITD) to EEG signals for automated seizure prediction

Intrinsic time-scale decomposition (ITD) is a new nonlinear method of time-frequency representation which can decipher the minute changes in the nonlinear EEG signals. In this work, we have automatically classified normal, interictal and ictal EEG signals using the features derived from the ITD representation. The energy, fractal dimension and sample entropy features computed on ITD representation coupled with decision tree classifier has yielded an average classification accuracy of 95.67%, sensitivity and specificity of 99% and 99.5%, respectively using 10-fold cross validation scheme. With application of the nonlinear ITD representation, along with conceptual advancement and improvement of the accuracy, the developed system is clinically ready for mass screening in resource constrained and emerging economy scenarios

MMP-9 expression in ocular surface tissues.

<p>Representative light micrographs of tissue sections showing MMP-9 expression in (A–D) corneal epithelium, (E–H) stromal keratocytes, (I–L) bulbar conjunctival epithelium, (M–P) lacrimal gland and (Q–T) meibomian gland of unwounded control eyes collected prior to surgery, and wounded eyes at various stages of healing. Arrows indicate stromal keratocytes. Arrow head shows an example of a conjunctival goblet cell.</p