Longitudinal Evaluation of Retinal Capillaries in Experimental Glaucoma

Purpose: Glaucoma is a leading cause of irreversible blindness worldwide. The loss of radial peripapillary capillaries (RPCs) has been hypothesized to play a role in retinal ganglion cell axon degeneration in glaucoma. Previous histological work has shown that structural alterations and losses of RPCs are associated with glaucomatous pathology. However, the lack of longitudinal in vivo data characterizing RPC alterations in glaucomatous eyes has limited understanding of how changes in RPCs relate with axonal changes during disease progression. The experiments described in this work were designed to (1) develop robust methods of imaging and analyzing RPCs in vivo and (2) investigate how changes in RPC perfusion relate to changes in the retinal nerve fiber layer (RNFL) over time in a non-human primate (NHP) model of experimental glaucoma (EG). Methods: First, the longitudinal repeatability of quantifying manually segmented RPCs acquired using adaptive optics scanning laser ophthalmoscope (AOSLO) spit detector imaging was evaluated in healthy NHP and human eyes in two or more sessions separated by ≥ 2 weeks. Second, a convolutional neural network (CNN) was developed to automatically segment perfused RPCs in AOSLO images. Third, perfused RPCs were imaged and quantified (using the developed CNN) approximately every 2 weeks after inducing unilateral EG in 9 rhesus monkeys. The RNFL and optic nerve head structure were imaged using spectral domain optical coherence tomography and quantified at each time point. Results: No significant differences were found in any examined capillary metric between sessions across healthy monkey and human eyes (P>.05; Mann-Whitney). The developed CNN objectively segments perfused capillaries with high accuracy (0.938). Changes in RPC perfusion occurred prior to RNFL thinning in 6 of 9 EG eyes. RNFL thinning occurred prior to a change in RPC perfusion in 2 EG eyes, while one EG eye had simultaneous changes in RNFL thickness and RPC perfusion. Conclusions: AOSLO imaging can be used to provide repeatable measures of perfused RPCs. The longitudinal data suggest that vascular factors may make some eyes more susceptible to axonal loss in early stages of the disease. The methods developed here may be applied to other retinal and optic nerve pathologies.Biomedical Engineering, Department o

Topical application of cannabinoid-ligands ameliorates experimental dry-eye disease

Purpose: Dry eye disease (DED) is a multifactorial disease, with limitations regarding efficacy and tolerability of applied substances. Among several candidates, the endocannabinoid system with its receptors (CB1R and CB2R) were reported to modulate inflammation, wound healing and pain, which are also core DED pathomechanisms. This study is to investigate the therapeutic responses of Delta-9 tetrahydrocannabinol (a non-selective agonist) and two selective antagonists, SR141716A (CB1R antagonist) and SR144528 (CB2R antagonist), as a topical application using a DED mouse model. Method: Experimental DED was induced in naive C57BL/6 mice. Expression of CBR at the ocular surface of naive and DED mice was determined by qPCR and in-situ hybridization. Either THC or CBR antagonists were compounded in an aqueous solution and dosed during the induction of DED. Tear production, cornea sensitivity, and cornea fluorescence staining were tested. At the end of each experiment, corneas were stained with beta 3-tubulin for analysis of corneal nerve morphology. Conjunctiva was analyzed for CD4(+) and CD8(+) infiltration. Results: CB1R and CB2R are present at the ocular surface, and desiccating stress increased CBR expressions (p < 0.05). After 10 days of DED induction, treated groups demonstrated a reduced CBR expression in the cornea, which was concurrent with improvements in the DED phenotype including fluorescence staining & inflammation. Applying THC protected corneal nerve morphology, thus maintained corneal sensitivity and reduced CD4(+) T-cell infiltration. The CB1R antagonist maintained cornea sensitivity without changing nerve morphology. Conclusions: Endocannabinoid receptor modulation presents a potential multi-functional therapeutic approach for DED

Cystathionine beta-synthase as novel endogenous regulator of lymphangiogenesis via modulating VEGF receptor 2 and 3

Lymphangiogenesis is a key player in several diseases such as tumor metastasis, obesity, and graft rejection. Endogenous regulation of lymphangiogenesis is only partly understood. Here we use the normally avascular cornea as a model to identify endogenous regulators of lymphangiogenesis. Quantitative trait locus analysis of a large low-lymphangiogenic BALB/cN x high-lymphangiogenic C57BL/6 N intercross and prioritization by whole-transcriptome sequencing identify a novel gene responsible for differences in lymphatic vessel architecture on chromosome 17, the cystathionine beta-synthase (Cbs). Inhibition of CBS in lymphatic endothelial cells results in reduce proliferation, migration, altered tube-formation, and decrease expression of vascular endothelial growth factor (VEGF) receptor 2 (VEGF-R2) and VEGF-R3, but not their ligands VEGF-C and VEGF-D. Also in vivo inflammation-induced lymphangiogenesis is significantly reduce in C57BL/6 N mice after pharmacological inhibition of CBS. The results confirm CBS as a novel endogenous regulator of lymphangiogenesis acting via VEGF receptor 2 and 3-regulation and open new treatment avenues in diseases associated with pathologic lymphangiogenesis. Quantitative trait locus analysis of BALB/cN x C57BL/6 N intercross identifies cystathionine beta-synthase as a previously unknown regulator of lymphangiogenesis by affecting VEGF-R2/VEGF-R3 expression in lymphatic endothelial cells

Ocular Graft-versus-Host Disease in a Chemotherapy-Based Minor-Mismatch Mouse Model Features Corneal (Lymph-) Angiogenesis

Ocular graft-versus-host disease (oGVHD) is a fast progressing, autoimmunological disease following hematopoietic stem cell transplantation, leading to severe inflammation of the eye and destruction of the lacrimal functional unit with consecutive sight-threatening consequences. The therapeutic window of opportunity is narrow, and current treatment options are limited and often insufficient. To achieve new insights into the pathogenesis and to develop new therapeutic approaches, clinically relevant models of oGVHD are desirable. In this study, the ocular phenotype was described in a murine, chemotherapy-based, minor-mismatch GVHD model mimicking early-onset chronic oGVHD, with corneal epitheliopathy, inflammation of the lacrimal glands, and blepharitis. Additionally, corneal lymphangiogenesis was observed as part of oGVHD pathogenesis for the first time, thus opening up the investigation of lymphangiogenesis as a potential therapeutic and diagnostic tool