Aqueous humor analysis to assess ocular environment

Aqueous is intimately related to the cells of anterior and posterior chambers, which affect its composition. Aqueous analysis can provide useful information regarding physiological and pathophysiological processes in the eye. Using multiplex analysis we found that out of 90 analytes evaluated, 52 (57%) were detectable in the normal aqueous. To place these results in biological context, the functional pathways, networks, biological processes and disease processes these analytes represented were identified. Numbers of ocular pathology related processes were represented in aqueous. The detected analytes represented biomarkers of a number of relevant disease processes, including vascular diseases, arteriosclerosis, ischemia, necrosis and inflammation. To provide the proof of principle that aqueous profile could offer useful information about the pathophysiological processes, we analyzed 2 aqueous samples from diabetic patients. There were differences in normal and diabetic samples, including those relevant to diabetic retinopathy such as VEGF, C-reactive protein, glutathione and cytokines. Several biomarker groups for disease processes relevant to diabetes were perturbed. Moreover, ocular pathology/pathophysiology specific MAPs can be developed and used to analyze aqueous.

Implications of anti-VEGF therapy on ocular physiology and pathobiology

Vascular endothelial growth factor (VEGF) plays a key role in neovascularization by stimulating proliferation and migration of vascular endothelial cells. Anti-VEGF therapy, bevacizumab, acts by binding to VEGF and preventing its effects. However, this linear interaction represents only a partial view of pathobiology of neovascular diseases and the anti-VEGF treatment. To obtain an integrated view of the processes involved in ocular neovascularization we applied systems approach and investigated whether intravitreal bevacizumab injection had a global effect in normalizing the ocular physiology perturbed by the disease. Our results show intraocular bevacizumab injections changes the perturbed physiological environment of the eye towards normalization. Its effects reached beyond neutralizing VEGF. The results also demonstrate that large scale analysis of aqueous using systems approach could provide useful insight regarding ocular disease, their pathophysiology and treatment responses.

Although anti-VEGF therapy is widely used for ocular neovascular disorders, its safety has not been well studied. We looked at the safety profile of commonly used doses of bevacizumab on various retinal cells and found it to be safe.

Bevacizumab can also be potentially used for corneal neovascularization. Our studies show that bevacizumab is non toxic to corneal epithelium and the corneal fibroblasts in concentrations commonly used for ani-angiogenic purposes.

However, we found that bevacizumab may stimulate the proliferation nonspecifically by increasing the protein content of the cell growth environment. Results suggest that the typical amount of bevacizumab clinically injected into the vitreous can alter the internal milieu of the eye by increasing protein concentration that is sufficient to stimulate cell proliferation in certain cells. This might be especially relevant for cells not under VEGF control. Further studies are under way to determine if this could have any clinical implications.

Safety of use of various dyes in ocular surgery

Internal limiting membrane (ILM) peeling improves surgical outcomes in idiopathic macular hole, diabetic macular edema and epiretinal membrane. ILM, a thin transparent membrane is often difficult to visualize during vitreoretinal surgery and staining with vital dyes such as indocyanine green (ICG) improve visualization of the ILM intra operatively and facilitate its removal.

Recently, newer vital dyes have been proposed for staining internal limiting membrane, as an alternative to indocyanine green, which has been shown to be toxic to the retinal cells, in both in-vitro and clinical studies. Our experimental studies evaluated the comparative toxicities of new vital dyes — bromophenol blue, brilliant blue G and infracyanine green (IfCG) to establish a safe alternative to ICG.

In this in-vitro study, we report that among the 3 dyes tested, IfCG is the least toxic and a safe alternative to ICG for staining ICG during vitrectomy.