Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

University College London Institue of Ophthalmology

Moorfields Eye Hospital

We investigated the cellular mechanisms underlying force generation and matrix contraction, using human corneal, Tenon's and scleral fibroblasts in a standard collagen matrix. We used timelapse light and confocal reflection microscopy to analyse concomitantly cell behaviour and matrix remodeling during contraction and devised a novel index to quantify dynamic cell behaviour in 3D. Based on the previously described culture force monitor, a novel simultaneous imaging and micro-culture force monitor system (SIM-CFM) was developed to measure the mechanical strain generated during matrix contraction whilst simultaneously recording cell and matrix behaviour. Ocular fibroblasts show marked differences in macroscopic matrix contraction profiles, with corneal fibroblasts inducing the strongest, and scleral the weakest, contraction. We identified four factors that determine the early matrix contraction profile: 1) cell size, 2) intrinsic cellular force, 3) dynamic cell protrusive activity and 4) net pericellular matrix displacement. Intrinsic cellular force and dynamic activity appear to be independent unique characteristics of each cell type and might serve as predictors of matrix contraction. The identification of these factors raises the fundamental new possibilities of predicting the ability of tissues to contract and scar and of modulating tissue contraction by targeting intracellular pathways linked to protrusive activity and force generation.

Dynamic protrusive cell behaviour generates force and drives early matrix contraction by fibroblasts.

Following all types of glaucoma filtration surgery (GFS), scarring still poses the major threat to long-term success. The healing and scarring determine the percentage of patients achieving low final intraocular pressures (IOPs) that are associated with virtually no glaucoma progression. The use of antifibrotic agents to inhibit scarring of trabeculectomy blebs is now a well-established clinical practice. Unfortunately, severe complications such as leakage, infection, hypotony, and endophthalmitis with complete loss of vision may occur. In addition, surgery still fails in some individuals despite maximal doses of current antifibrotics. Better therapeutic agents are needed. Many promising new agents are being evaluated clinically and in vitro. In this chapter, we will discuss our current understanding of the wound healing process after glaucoma surgery and promising new treatment modalities.

Modulation of wound healing during and after glaucoma surgery.

The origins and insertions of rectus muscles: Paul J. Tillaux and Charles B. Lockwood.

P. J. Tillaux: of the way the muscles insert onto the sclera. 1877.

Conjunctival and episcleral bleeding can interfere with visualization of anatomic structures during strabismus surgery. In the United Kingdom, dilute adrenaline (epinephrine) is routinely used to induce vasoconstriction at the beginning of strabismus surgery. Because it causes mydriasis and mildly inhibits accommodation, it is relatively contraindicated if early postoperative suture adjustment is planned.

Brimonidine and apraclonidine as vasoconstrictors in adjustable strabismus surgery.

The 3 most common causes of visual impairment and legal blindness in developed countries (age-related macular degeneration, glaucoma, and diabetic retinopathy) share 1 end point: the loss of neural cells of the eye. Although recent treatment advances can slow down the progression of these conditions, many individuals still suffer irreversible loss of vision. Research is aimed at developing new treatment strategies to rescue damaged photoreceptors and retinal ganglion cells (RGC) and to replace lost cells by transplant. The neuroprotective and regenerative potential of stem and progenitor cells from a variety of sources has been explored in models of retinal disease and ganglion cell loss. Continuous intraocular delivery of neurotrophic factors via stem cells (SC) slows down photoreceptor cells and RGC loss in experimental models. Following intraocular transplantation, SC are capable of expressing proteins and of developing a morphology characteristic of photoreceptors or RGC. Recently, recovery of vision has been achieved for the first time in a rodent model of retinal dystrophy, using embryonic SC differentiated into photoreceptors prior to transplant. This indicates that clinically significant synapse formation and acquisition of the functional properties of retinal neurons, and restoration of vision, are distinct future possibilities.

Current approaches and future prospects for stem cell rescue and regeneration of the retina and optic nerve.

Although fibroblasts play an essential part during the wound healing response, the mechanisms by which they mediate tissue remodelling and contraction are still unclear. Using live cell and matrix imaging within 3D free-floating fibroblast-populated collagen lattices as a model for tissue contraction, we compared the behaviour of a range of fibroblasts with low and high contraction abilities and analysed the effect of the broad spectrum MMP-inhibitor GM6001 on cell behaviour and matrix contraction. We identified two mechanisms underlying matrix contraction, one via direct cell-mediated contractile activity, the second through matrix degradation. These appear to be linked to cell morphology and regulated by the collagen concentration within the matrix. Cells with a rounded morphology proliferated in the matrix but did not remodel it efficiently, resulting in a poor ability to contract matrices. Cells with an elongated morphology showed higher levels of protrusive activity, leading to efficient matrix remodelling and contraction. GM6001 inhibited week-long matrix contraction to various extents with the different cell lines. However, quantitative analysis of the cell protrusive activity showed that GM6001 consistently decreased cell dynamics in 3D by about 20%, and this was correlated with a significant reduction in early matrix contraction. Overall our results suggest that although fibroblast-mediated matrix contraction depends on both cell dynamics and MMP-mediated matrix degradation, the efficiency of GM6001 treatment in preventing contraction might be linked to a direct effect on cell dynamics.

The effect of MMP inhibitor GM6001 on early fibroblast-mediated collagen matrix contraction is correlated to a decrease in cell protrusive activity.

To develop and validate a novel ex vivo model of conjunctival contraction.

Advancing the treatment of conjunctival scarring: a novel ex vivo model.

Previous work has suggested a major role of scleral biomechanics in the pathogenesis of glaucoma. Since fiber orientation in connective tissues is a key determinant of tissue biomechanics, experimental characterization of scleral fiber orientation is needed to fully understand scleral biomechanics. This is a report of baseline experimental measurements of fiber orientation in whole normal rat scleras.

Quantitative mapping of scleral fiber orientation in normal rat eyes.

Vascular endothelial growth factor A (VEGF-A) is a validated therapeutic target in several angiogenic- and vascular permeability-related pathological conditions, including certain cancers and potentially blinding diseases, such as age-related macular degeneration and diabetic retinopathy. We and others have shown that VEGF-A also plays an important role in neuronal development and neuroprotection, including in the neural retina. Antagonism of VEGF-A function might therefore present a risk to neuronal survival as a significant adverse effect. Herein, we demonstrate that VEGF-A acts directly on retinal ganglion cells (RGCs) to promote survival. VEGF receptor-2 signaling via the phosphoinositide-3-kinase/Akt pathway was required for the survival response in isolated RGCs. These results were confirmed in animal models of staurosporine-induced RGC death and experimental hypertensive glaucoma. Importantly, we observed that VEGF-A blockade significantly exacerbated neuronal cell death in the hypertensive glaucoma model. Our findings highlight the need to better define the risks associated with use of VEGF-A antagonists in the ocular setting.

VEGF-A is necessary and sufficient for retinal neuroprotection in models of experimental glaucoma.

To test agreement of two methods to measure intraocular pressure (IOP): rebound tonometry (RBT) and gold standard Goldmann applanation tonometry (GAT) in children with glaucoma.

Comparison of handheld rebound tonometry with Goldmann applanation tonometry in children with glaucoma: a cohort study.

Evidence-based medicine requires primary results from randomized controlled trials and high-quality observational studies; however, in pediatric medicine it can be difficult to enroll sufficient numbers of children to reach sample sizes required for statistical significance. The experience of clinicians and researchers with the practicalities of recruitment has not been explored, but could potentially inform best practice and lead to the development of successful research networks. Perceived barriers and facilitators to recruitment in child eye health in the United Kingdom, where adoption of trials and studies onto a central database has created a register of high quality projects, were investigated.

Annegret Dahlmann-Noor

University College London Institue of Ophthalmology

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres