Search

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Starting in 2021, Poland's emission reduction target for 2030, in the sectors not covered by the European Union Emissions Trading System (EU ETS) is −7% compared to the emissions level in the non-ETS sectors in 2005. This is a big change in comparison with the target for the period of 2013–2020, which assumed potential emissions increase relative to the same baseline and was +14% by 2020. In order to cope with the need to make a much greater reduction effort, Poland will likely need to use flexibility mechanisms to support achieving the more ambitious target.

In the paper we consider the problem of identification of large-scale interconnected systems. Accurate models of complex nets are needed especially for optimal control in production and transportation systems. The specifics lay in the fact that individual elements cannot be disconnected and excited by arbitrary input processes for identification purposes. Moreover, structural interactions cause correlations between interaction signals. In particular, any output random disturbances can be transferred into the other inputs. It leads to cross-correlation problems, very difficult from the effective modelling point of view. First attempts made in 1980's were limited to static linear blocks, and in practice the results were rather devoted to linear dynamic systems working in steady state. In this paper we generalize the approach for components, which are both dynamic and nonlinear. All blocks are represented by two-channel Hammerstein systems (used e.g. in modelling of real heating processes). The least squares estimate is applied to identify unknown parameters of a system. The parameters of particular elements are obtained in singular value decomposition procedure. The algorithm as a whole is illustrated in simple simulation example. [ABSTRACT FROM AUTHOR]

Intraocular pressure (IOP) elevation is the primary risk factor and currently the main treatable factor for progression of glaucomatous optic neuropathy. In addition to direct clinical and living animal in vivo studies, ex vivo perfusion of anterior segments and whole eyes is a key technique for studying conventional outflow function as it is responsible for IOP regulation. We present well-tested experimental details, protocols, considerations, advantages, and limitations of several ex vivo model systems for studying IOP regulation. These include: (1) perfused whole globes, (2) stationary anterior segment organ culture, (3) perfused human anterior segment organ culture, (4) perfused animal anterior segment organ culture, (5) perfused human corneal rims, and (6) perfused human anterior segment wedges. These methods, with due consideration paid to their strengths and limitations, comprise a set of very strong tools for extending our understanding of IOP regulation.

Purpose: Loss-of-function variants in the ANGPTL7 gene are associated with protection from glaucoma and reduced intraocular pressure (IOP). We investigated the role of ANGPTL7 in IOP homeostasis and its potential as a target for glaucoma therapeutics., Methods: IOP, outflow facility, and outflow tissue morphology of Angptl7 knockout (KO) mice were assessed with and without dexamethasone (Dex). ANGPTL7 was quantified in conditioned media from human trabecular meshwork cells in response to Dex, in effluent from perfused human donor eyes, and in aqueous humor from human patients treated with steroids. Antibodies to ANGPTL7 were generated and tested in three-dimensional (3D) culture of outflow cells and perfused human donor eyes. Rabbits were injected intravitreally with a neutralizing antibody targeting ANGPTL7, and IOP was measured., Results: IOP was significantly elevated, but outflow facility and outflow tissue morphology were not different between Angptl7 KO mice and littermates. When challenged with Dex, IOP increased in wild-type but not Angptl7 KO mice. In human samples, increased ANGPTL7 was seen in the aqueous humor of patients treated with steroids, regardless of glaucoma status. Using 3D culture, recombinant ANGPTL7 decreased, and ANGPTL7-blocking antibodies increased hydraulic conductivity. Significantly, outflow facility increased in human eyes treated ex vivo with ANGPTL7-blocking antibodies, and IOP decreased for 21 days in rabbits after a single injection of blocking antibodies., Conclusions: Using multiple models, we have demonstrated that excess ANGPTL7 increases outflow resistance and IOP and that neutralizing ANGPTL7 has beneficial effects in both naïve and steroid-induced hypertensive eyes, thus motivating the development of ANGPTL7-targeting therapeutics for the treatment of glaucoma.

Ocular hypertension is caused by dysregulated outflow resistance regulation by the conventional outflow (CO) pathway. The physiology of the CO pathway can be directly studied during ex vivo ocular perfusions. In addition to measuring outflow resistance generation by the CO tissues, perfusion media that is conditioned by CO pathway cells can be collected upon exiting the eye as effluent. Thus, contents of effluent include factors contributed by upstream cells that report on the (dys)functionality of the outflow tissues. Two methods have been used in the past to monitor effluent contents from perfused eyes, each with their limitations. To overcome these limitations, we designed and printed a metabolic chamber to accommodate eyes of different sizes during perfusions. To test this new chamber, human eyes were perfused for 4 h at constant flow rate of 2.5 μl/min, while pressure was continuously monitored and effluent was collected every hour. Facility was 0.28 ± 0.16 μl/min/mmHg for OD eyes and 0.33 ± 0.11 μl/min/mmHg for OS eyes (n = 3). Effluent samples were protein rich, with protein concentration ranging from 2700 to 10,000 μg/ml for all eyes and timepoints (N = 3). Effluent samples expressed proteins that were actively secreted by the TM and easily detectible including MYOC and MMP2. Taken together, our model provides a reliable method to collect effluent from ex vivo human eyes, while maintaining whole globe integrity., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Primary open-angle glaucoma is one of the leading causes of blindness worldwide. With limited therapeutics targeting the pathogenesis at the trabecular meshwork (TM), there is a great need for identifying potential new targets. Recent evidence has implicated Toll-like receptor 4 (TLR4) and it is signaling pathway in augmenting the effects of transforming growth factor beta-2 (TGFβ2) and downstream extracellular matrix production. In this review, we examine the role of TLR4 signaling in the trabecular meshwork and the interplay between endogenous activators of TLR4 (damage-associated molecular patterns (DAMPs)), extracellular matrix (ECM), and the effect on intraocular pressure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mzyk, Hernandez, Le, Ramirez and McDowell.)

Although the extracellular matrix (ECM) in trabecular meshwork (TM) cells is known to be important in intraocular pressure (IOP) regulation, the molecular mechanisms involved in generating a glaucomatous environment in the TM are not completely understood. Recently we identified a molecular pathway, transforming growth factor beta 2 (TGFβ2)-toll-like receptor 4 (TLR4) signaling crosstalk, as an important regulator of glaucomatous damage in the TM, which contributes to fibrosis. Here we evaluate a novel molecular target, A20, also known as tumor necrosis factor alpha-induced protein 3 (TNFAIP3), which may help to block pathological TGFβ2-TLR4 signaling. Primary human TM cells were analyzed for A20 message and for A20 and fibronectin protein expression after treatment with TGFβ2. A20 message increased when the TLR4 pathway was inhibited in TM cells. In addition, TGFβ2, a known inducer of fibrosis, increased fibronectin expression, while at the same time decreasing the expression of A20. We then overexpressed A20 in TM cells in order to test the effect on treatment with TGFβ2, lipopolysaccharide (LPS), or cellular fibronectin extra domain A (cFN-EDA). Importantly, overexpression of A20 rescued the fibrotic response when TM cells were treated with TGFβ2, LPS, or cFN-EDA. In situ hybridization was used to probe for A20 RNA expression in age-matched control (C57BL/6J) mice and mice that constitutively express the EDA isoform of fibronectin (B6.EDA +/+ ). In this novel mouse model of glaucoma, A20 RNA was increased versus age-matched control mice in a cyclic manner at 6 weeks and 1 year of age, but not at 8 months. Overall, these data suggest that A20 may work through a negative feedback mechanism attenuating the ability of TGFβ2-TLR4 signaling to induce fibrosis.

The optic nerve head (ONH) is a place of vulnerability during glaucoma progression due to increased intraocular pressure damaging the retinal ganglion cell axons. The molecular signaling pathways involved in generating glaucomatous ONH damage has not been fully elucidated. There is a great deal of evidence that pro-fibrotic TGFβ2 signaling is involved in modulating the ECM environment within the lamina cribrosa (LC) region of the ONH. Here we investigated the role of signaling crosstalk between the TGFβ2 pathway and the toll-like receptor 4 (TLR4) pathway within the LC. ECM deposition was examined between healthy and glaucomatous human ONH sections, finding increases in fibronectin and fibronectin extra domain A (FN-EDA) an isoform of fibronectin known to be a damage associated molecular pattern (DAMP) that can activate TLR4 signaling. In human LC cell cultures derived from healthy donor eyes, inhibition of TLR4 signaling blocked TGFβ2 induced FN and FN-EDA expression. Activation of TLR4 by cellular FN (cFN) containing the EDA isoform increased both total FN production and Collagen-1 production and this effect was dependent on TLR4 signaling. These studies identify TGFβ2-TLR4 signaling crosstalk in LC cells of the ONH as a novel pathway regulating ECM and DAMP production., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Purpose: Intracellular iron trafficking and the characteristics of iron distribution from different sources are poorly understood. We previously determined that the lens removes excess iron from fluids of inflamed eyes. In the current study, we examined uptake and intracellular distribution of ⁵⁹Fe from iron transport protein transferrin or ferric chloride (nontransferrin-bound iron [NTBI]) in cultured canine lens epithelial cells (LECs). Because lens tissue physiologically functions under low oxygen tension, we also tested effects of hypoxia on iron trafficking. Excess iron, not bound to proteins, can be damaging to cells due to its ability to catalyze formation of reactive oxygen species., Methods: LECs were labeled with ⁵⁹Fe-Tf or ⁵⁹FeCl₃ under normoxic or hypoxic conditions. Cell lysates were fractioned into mitochondria-rich, nuclei-rich, and cytosolic fractions. Iron uptake and its subcellular distribution were measured by gamma counting., Results: ⁵⁹Fe accumulation into LECs labeled with ⁵⁹Fe-Tf was 55-fold lower as compared with that of ⁵⁹FeCl₃. Hypoxia (24 hours) decreased uptake of iron from transferrin but not from FeCl₃. More iron from ⁵⁹FeCl₃ was directed to the mitochondria-rich fraction (32.6%-47.7%) compared with ⁵⁹Fe from transferrin (10.6%-12.6%). The opposite was found for the cytosolic fraction (8.7%-18.3% and 54.2%-46.6 %, respectively). Hypoxia significantly decreased iron accumulation in the mitochondria-rich fraction of LECs labeled with ⁵⁹Fe-Tf ., Conclusions: There are source-dependent differences in iron uptake and trafficking. Uptake and distribution of NTBI are not as strictly regulated as that of iron from transferrin. Excessive exposure to NTBI, which could occur in pathological conditions, may oxidatively damage organelles, particularly mitochondria.