The ex vivo human translaminar autonomous system to study spaceflight associated neuro-ocular syndrome pathogenesis.

Spaceflight-Associated Neuro-ocular Syndrome (SANS) is a significant unexplained adverse reaction to long-duration spaceflight. We employ an ex vivo translaminar autonomous system (TAS) to recreate a human ocular ground-based spaceflight analogue model to study SANS pathogenesis. To recapitulate the human SANS conditions, human ocular posterior segments are cultured in the TAS model for 14 days. Translaminar pressure differentials are generated by simulating various flow rates within intracranial pressure (ICP) and intraocular (IOP) chambers to maintain hydrostatic pressures of ICP: IOP (12:16, 15:16, 12:21, 21:16 mmHg). In addition, optic nerves are mechanically kinked by 6- and 10-degree tilt inserts for the ICP: IOP;15:16 mmHg pressure paradigm. The TAS model successfully maintains various pressure differentials for all experimental groups over 14 days. Post culture, we determine inflammatory and extracellular component expression changes within posterior segments. To further characterize the SANS pathogenesis, axonal transport capacity, optic nerve degeneration and retinal functional are measured. Identifiable pathogenic alterations are observed in posterior segments by morphologic, apoptotic, and inflammatory changes including transport and functional deficits under various simulated SANS conditions. Here we report our TAS model provides a unique preclinical application system to mimic SANS pathology and a viable therapeutic testing device for countermeasures.

Fibronectin extra domain A (FN-EDA) elevates intraocular pressure through Toll-like receptor 4 signaling.

Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage, which leads to impaired aqueous humor outflow. Here, we explore a novel molecular mechanism involved in glaucomatous TM damage. We investigated the role of an endogenous Toll-like receptor 4 (TLR4) ligand, fibronectin-EDA (FN-EDA), in TGFß2-induced ocular hypertension in mice. We utilized transgenic mouse strains that either constitutively express only FN containing the EDA isoform or contain an EDA-null allele and express only FN lacking EDA, with or without a mutation in Tlr4, in our inducible mouse model of ocular hypertension by injection of Ad5.TGFß2. IOP was measured over time and eyes accessed by immunohistochemistry for total FN and FN-EDA expression. Constitutively active EDA caused elevated IOP starting at 14 weeks of age. Ad5.TGFß2 induced ocular hypertension in wildtype C57BL/6J mice and further amplified the IOP in constitutively active EDA mice. TLR4 null and EDA null mice blocked Ad5.TGFß-induced ocular hypertension. Total FN and FN-EDA isoform expression increased in response to Ad5.TGFß2. These data suggest that both TLR4 and FN-EDA contribute to TGFß2 induced ocular hypertension.

Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments.

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular pressure (IOP) which can identify various pathogenic paradigms related to the glaucoma pathogenesis. Ex vivo human anterior segment perfusion organ culture models have previously been successfully utilized and applied as effective technologies for the discovery of glaucoma pathogenesis and testing of therapeutics. Preclinical drug screening and research performed on ex vivo human organ systems can be more translatable to clinical research. This article describes in detail the generation and operation of a novel ex vivo human translaminar pressure model called the translaminar autonomous system (TAS). The TAS model can independently regulate ICP and IOP using human donor posterior segments. The model allows for studying pathogenesis in a preclinical manner. It can reduce the use of living animals in ophthalmic research. In contrast to in vitro experimental models, optic nerve head (ONH) tissue structure, complexity, and integrity can also be maintained within the ex vivo TAS model.

Effects of Toll-Like Receptor 4 Inhibition on Transforming Growth Factor-ß2 Signaling in the Human Trabecular Meshwork.

Purpose: Transforming growth factor-ß2 (TGFß2) and Toll-like receptor 4 (TLR4) crosstalk have been implicated in extracellular matrix regulation in the trabecular meshwork (TM) and ocular hypertension in mice. We investigated TLR4 expression in normal and glaucomatous human trabecular meshwork (HTM) sections and utilized a human perfusion organ culture model to determine TGFß2-TLR4 signaling crosstalk in glaucoma. Methods: Expression of TLR4 was determined in TM of normal and glaucomatous human eyes. Anterior segments of paired human eyes were perfused at a constant flow rate (2.5 µL/min) for 4 days to acquire stable baseline intraocular pressures (IOPs). We treated paired eyes with two different treatment paradigms: (1) TGFß2 in one eye and vehicle control in the paired eye, (2) TGFß2 in one eye and TGFß2 + TLR4 inhibitor TAK-242 in the paired eye. Perfusate and TM tissue were collected and analyzed for fibronectin (FN) and collagen IV (COLIV) expression. Results: We observed increased TLR4 expression in glaucomatous HTM sections compared to normal (age-matched) (P < 0.05). Significant elevation of IOP was detected in 47% of TGFß2-treated anterior segments (P < 0.01) compared to control, and in TGFß2 treated compared with co-treatment with TGFß2 + TLR4 inhibitor (P < 0.0001). An increase in FN and COLIV expression was observed after TGFß2 treatment, and inhibition of TLR4 signaling decreased TGFß2-induced FN and COLIV expression in perfusate (P < 0.05). Conclusions: These studies identify TGFß2-TLR4 crosstalk as a novel pathway in glaucoma. They provide a potential new target to lower IOP and explore glaucoma pathogenesis.

BMP and Activin Membrane Bound Inhibitor Regulates the Extracellular Matrix in the Trabecular Meshwork.

Purpose: The trabecular meshwork (TM) has an important role in the regulation of aqueous humor outflow and IOP. Regulation of the extracellular matrix (ECM) by TGFß2 has been studied extensively. Bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) has been shown to inhibit or modulate TGFß2 signaling. We investigate the role of TGFß2 and BAMBI in the regulation of TM ECM and ocular hypertension. Methods: Mouse TM (MTM) cells were isolated from B6;129S1-Bambitm1Jian/J flox mice, characterized for TGFß2 and dexamethasone (DEX)-induced expression of fibronectin, collagen-1, collagen-4, laminin, α-smooth muscle actin, cross-linked actin networks (CLANs) formation, and DEX-induced myocilin (MYOC) expression. MTM cells were transduced with Ad5.GFP to identify transduction efficiency. MTM cells and mouse eyes were transduced with Ad5.Null, Ad5.Cre, Ad5.TGFß2, or Ad5.TGFß2 + Ad5.Cre to evaluate the effect on ECM production, IOP, and outflow facility. Results: MTM cells express TM markers and respond to DEX and TGFß2. Ad5.GFP at 100 MOI had the highest transduction efficiency. Bambi knockdown by Ad5.Cre and Ad5.TGFß2 increased fibronectin, collagen-1, and collagen-4 in TM cells in culture and tissue. Ad5.Cre, Ad5.TGFß2, and Ad5.TGFß2 + Ad5.Cre each significantly induced ocular hypertension and lowered aqueous humor outflow facility in transduced eyes. Conclusions: We show for the first time to our knowledge that knockdown of Bambi alters ECM expression in cultured cells and mouse TM, reduces outflow facility, and causes ocular hypertension. These data provide a novel insight into the development of glaucomatous TM damage and identify BAMBI as an important regulator of TM ECM and ocular hypertension.