Human Glaucoma Research Articles

Retinal Ganglion Cell Subtypes and Their Vulnerability in Glaucoma.

The detection of selective retinal ganglion cell damage in glaucoma has been a long sought-after goal, not just for the development of clinical tests for the early detection of glaucoma but for the elucidation of potential mechanisms underlying retinal ganglion cell loss. Early reports of the selective vulnerability of larger retinal ganglion cells (RGCs) in human studies did not translate simply to the loss of a particular class of RGC but more likely reflected shrinkage and degeneration across all RGC classes. Subsequent studies of nonhuman primate (NHP) models of glaucoma indicated some selectivity with great damage to the magnocellular vs parvocellular pathways. More recently, rodent models of experimental glaucoma have highlighted a selective vulnerability of OFF-centered RGCs-particularly those with transient responses. Selectivity for OFF pathway damage is also seen as a trend in a rat model of glaucoma. These data support the concept that some RGCs are more vulnerable to the effects of glaucoma damage. This chapter covers some of the methods to elucidate RGC damage and the relevance of model selection to mimic human glaucoma rather than just RGC death.

Trabecular Meshwork Abnormalities in a Model of Congenital Glaucoma Due to LTBP2 Mutation.

To characterize early trabecular meshwork (TM) morphologic abnormalities in a feline model of human primary congenital glaucoma (PCG) caused by mutation in LTBP2. Eyes from 41 cats, including 19 normal and 22 homozygous for LTBP2 mutation, across various postnatal stages (birth, 2 weeks, 5 weeks, and 12 weeks) were paraformaldehyde fixed, anterior segments dissected, post-fixed in glutaraldehyde, osmicated, and processed and sectioned for transmission electron microscopy. Cell morphology, nuclear shape, and intertrabecular space (ITS) were quantitatively assessed, and the structure of the fibrillar extracellular matrix in the TM was systematically evaluated. The earliest differences in TM morphology between PCG and normal cats were identified at 2 weeks postnatally. Elastic fibers in the TM were discontinuous and disorganized (P = 0.0122), and by 5 weeks of age PCG cats presented significantly less ITS (P = 0.0076) and morphologically rounder TM cells than normal cats (P = 0.0293). By 12 weeks of age, the ITS was further collapsed (P < 0.0001), and the TM cells were morphologically elongated and attenuated in PCG compared to controls (P = 0.0028). In this feline model of PCG due to LTBP2 mutation, development of ultrastructural TM extracellular matrix abnormalities are first observed by 2 weeks and cellular abnormalities by 5 weeks of age. By 12 weeks of age, when intraocular pressure becomes significantly elevated, the TM morphologic abnormalities are already well established. These findings suggest that the postnatal period between 0 and 5 weeks of age is critical for TM and PCG development and progression in cats.

Transcriptomic profiling of Schlemm’s canal cells reveals a lymphatic-biased identity and three major cell states

Schlemm’s canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm’s canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6 J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4,500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6 J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize three molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states.

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6 J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4,500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6 J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize three molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

In vitro comparison of human and murine trabecular meshwork cells: implications for glaucoma research

The trabecular meshwork (TM) is crucial for regulating intraocular pressure (IOP), and its dysfunction significantly contributes to glaucoma, a leading cause of vision loss and blindness worldwide. Although rodents are commonly used as animal models in glaucoma research, the applicability of these findings to humans is limited due to the insufficient understanding of murine TM. This study aimed to compare primary human TM (hTM) and murine TM (mTM) cells in vitro to enhance the robustness and translatability of murine glaucoma models. In this in vitro study, we compared primary hTM and mTM cells under simulated physiological and pathological conditions by exposing both cell types to the glucocorticoid dexamethasone (DEX) and Transforming Growth Factor β (TGFB2), both of which are critical in the pathogenesis of several ophthalmological diseases, including glaucoma. Phagocytic properties were assessed using microbeads. Cells were analyzed through immunocytochemistry (ICC) and Western blot (WB) to evaluate the expression of extracellular matrix (ECM) components, such as Fibronectin 1 (FN1) and Collagen IV (COL IV). Filamentous-Actin (F-Act) staining was used to analyze cross-linked actin network (CLAN) formation. Additionally, we evaluated cytoskeletal components, including Vimentin (VIM), Myocilin (MYOC), and Actin-alpha-2 (ACTA2). Our results demonstrated significant similarities between human and murine TM cells in basic morphology, phagocytic properties, and ECM and cytoskeletal component expression under both homeostatic and pathological conditions in vitro. Both human and murine TM cells exhibited epithelial-to-mesenchymal transition (EMT) after exposure to DEX or TGFB2, with comparable CLAN formation observed in both species. However, there were significant differences in FN1 and MYOC induction between human and murine TM cells. Additionally, MYOC expression in hTM cells depended on fibronectin coating. Our study suggests that murine glaucoma models are potentially translatable to human TM. The observed similarities in ECM and cytoskeletal component expression and the comparable EMT response and CLAN formation support the utility of murine models in glaucoma research. The differences in FN1 and MYOC expression between hTM and mTM warrant further investigation due to their potential impact on TM properties. Overall, this study provides valuable insights into the species-specific characteristics of TM and highlights opportunities to refine murine models for better relevance to human glaucoma.

3D printed gelatin methacryloyl hydrogels for perfusion culture of human trabecular meshwork cells and glaucoma studies.

Glaucoma, a progressive eye disease leading to irreversible blindness, currently affects over 70 million people globally. Elevated intraocular pressure (IOP) is implicated in its development. IOP is carefully regulated by the trabecular meshwork (TM). However, studying TM behavior has been limited to traditional tissue culture studies or costly ex vivo cultures of animal and donor eyes. Developing novel functional TM models could enhance cell/tissue behavior understanding and aid therapeutic development for glaucoma. In this study, we 3D printed a simplified and reproducible model of the human TM (hTM) and studied hTM cell behavior under static and dynamic cultures. Gelatin Methacryloyl bioinks proved suitable for printing with viable and proliferative hTM cells expressing crucial marker genes in response to glucocorticoid induction. This, to our knowledge, is the first functional 3D printed hTM model and aims to facilitate TM research. Moreover, this easily reproducible model could also be applicable in the study of numerous other cell types throughout the body.

Identification of Retinal Amyloid-Beta in Ex-Vivo Human Glaucoma Eyes Using a Novel Ocular Tracer.

To characterize the presence of amyloid-beta (Aβ) in human glaucoma retina and to test identification of retinal Aβ using a novel fluorescent Aβ-binding small molecule (AMDX-2011). Post-mortem human eyes with (n=4) and without (n=4) glaucoma were acquired from an eye bank. Retinas were dissected, flat-mounted, and fixed. Using the flat-mounts, immunofluorescence was performed against Aβ, AMDX-2011 staining was conducted, and images were acquired using fluorescence microscopy. Fluorescence microscopy demonstrated presence of Aβ signal that co-localized with AMDX-2011 staining in glaucoma retina. Co-labeled puncta appeared in all quadrants of the retina, including retina temporal to the optic nerve. The puncta were mainly located within the inner layers of the retina. Glaucoma retinas had more co-labeled puncta than control retinas in all locations (P = 0.002-0.02). Co-labeled puncta were also larger in the superior quadrant of glaucoma compared to control retinas (P = 0.02). Aβ was detected in human glaucomatous retina, and its distribution was mapped. AMDX-2011 identification of Aβ may lead to future diagnostic tests aimed at detecting Aβ in glaucoma patients.

Methylene Blue Reduces Electroretinogram Distortion and Ganglion Cell Death in a Rat Model of Glaucoma.

Glaucoma is the second leading cause of blindness worldwide and is, in most cases, a consequence of elevated intraocular pressure (IOP), ultimately resulting in the death of retinal ganglion cells (RGCs). Current treatments are mostly focused on normalizing IOP, but we propose the additional use of neuroprotective agents, including methylene blue (MB), to block the loss of RGCs. Wistar rats were subjected to episcleral vein cauterization (EVC) in the left eye while the right eye was sham-operated. One week later, they were divided into two groups, which were injected with either 2.0 mg/kg MB or phosphate-buffered saline (PBS), twice a day, for 7 days. Fifteen days after surgery, rats were tested with scotopic electroretinography (ERG) or pattern electroretinography (PERG). After sacrifice, the number of RGCs and the thickness of the inner retina (IR) were evaluated both in the peripheral and central areas of the retina. Scotopic ERG showed a marked reduction (p < 0.0001) on the a- and b-wave amplitude and oscillatory potential (OP) complexity of the eyes subjected to EVC. These parameters were significantly (p < 0.01) restored by the application of MB. PERG indicated that EVC was responsible for a very significant decrease in N2 amplitude (p < 0.0001) and prolongation of N2 implicit time (p < 0.0001). Treatment with MB significantly restored N2 amplitude (p < 0.0001). In parallel with the ERG results, morphological analysis showed a significant loss of RGCs (p < 0.0001) and IR thickness (p < 0.0001) in both the peripheral and central retinas subjected to EVC, which was significantly prevented (p < 0.0001) by MB treatment. We have shown that MB treatment can be effective in preventing physiological and morphological hallmarks of optic neuropathy in a model of ocular hypertension, which faithfully recapitulates human open-angle glaucoma. Due to its high safety profile, this drug could therefore represent a new pharmacologic strategy to prevent vision loss in glaucoma patients.

Oral nicotinamide provides robust, dose-dependent structural and metabolic neuroprotection of retinal ganglion cells in experimental glaucoma

A compromised capacity to maintain NAD pools is recognized as a key underlying pathophysiological feature of neurodegenerative diseases. NAD acts as a substrate in major cell functions including mitochondrial homeostasis, cell signalling, axonal transport, axon/Wallerian degeneration, and neuronal energy supply. Dendritic degeneration is an early marker of neuronal stress and precedes cell loss. However, little is known about dendritic structural preservation in pathologic environments and remodelling in mature neurons. Retinal ganglion cell dendritic atrophy is an early pathological feature in animal models of the disease and has been demonstrated in port-mortem human glaucoma samples. Here we report that a nicotinamide (a precursor to NAD through the NAD salvage pathway) enriched diet provides robust retinal ganglion cell dendritic protection and preserves dendritic structure in a rat model of experimental glaucoma. Metabolomic analysis of optic nerve samples from the same animals demonstrates that nicotinamide provides robust metabolic neuroprotection in glaucoma. Advances in our understanding of retinal ganglion cell metabolic profiles shed light on the energetic shift that triggers early neuronal changes in neurodegenerative diseases. As nicotinamide can improve visual function short term in existing glaucoma patients, we hypothesize that a portion of this visual recovery may be due to dendritic preservation in stressed, but not yet fully degenerated, retinal ganglion cells.

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states.

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

Complement Factor B Inhibition or Deletion Is Not Sufficient to Prevent Neurodegeneration in a Murine Model of Glaucoma.

Purpose: Activation of the classical complement pathway is thought to contribute to the development and progression of glaucoma. The role of alternative complement or amplification pathways in glaucoma is not well understood. We evaluated complement factor B (FB) expression in postmortem human ocular tissues with or without glaucoma and the effect of FB inhibition and deletion in a mouse ocular hypertensive model of glaucoma induced by photopolymerized hyaluronic acid glycidyl methacrylate (HAGM). Methods: Human CFB mRNA in human eyes was assessed by RNAscope and TaqMan. HAGM model was performed on C57BL6/J mice. The effect of FB in HAGM model was evaluated with an oral FB inhibitor and Cfb-/- mice. Complement mRNA and proteins in mouse eyes were assessed by TaqMan and western blot, respectively. Results: CFB mRNA in human glaucomatous macular neural retina and optic nerve head was upregulated. Cfb mRNA is also upregulated in the HAGM model. Oral FB inhibitor, ED-79-GX17, dosed daily at 200 mg/kg for 3 days after intraocular pressure (IOP) induction in wild-type mice showed complement inhibition in ocular tissues and significantly inhibited systemic complement levels. Daily dosing of ED-79-GX17 for 30 days or Cfb deletion was also unable to prevent retinal ganglion cell or axon loss 30 days after IOP induction in mice. Conclusion: The alternative complement component FB may not substantially contribute to RGC loss in the HAGM mouse glaucoma model despite upregulation of Cfb expression and activation of the alternative pathway. The relevance of these findings to human glaucoma remains to be determined.

Remyelination-oriented clemastine treatment attenuates neuropathies of optic nerve and retina in glaucoma.

As one of the top causes of blindness worldwide, glaucoma leads to diverse optic neuropathies such as degeneration of retinal ganglion cells (RGCs). It is widely accepted that the level of intraocular pressure (IOP) is a major risk factor in human glaucoma, and reduction of IOP level is the principally most well-known method to prevent cell death of RGCs. However, clinical studies show that lowering IOP fails to prevent RGC degeneration in the progression of glaucoma. Thus, a comprehensive understanding of glaucoma pathological process is required for developing new therapeutic strategies. In this study, we provide functional and histological evidence showing that optic nerve defects occurred before retina damage in an ocular hypertension glaucoma mouse model, in which oligodendroglial lineage cells were responsible for the subsequent neuropathology. By treatment with clemastine, an Food and Drug Administration (FDA)-approved first-generation antihistamine medicine, we demonstrate that the optic nerve and retina damages were attenuated via promoting oligodendrocyte precursor cell (OPC) differentiation and enhancing remyelination. Taken together, our results reveal the timeline of the optic neuropathies in glaucoma and highlight the potential role of oligodendroglial lineage cells playing in its treatment. Clemastine may be used in future clinical applications for demyelination-associated glaucoma.

Glaucoma and the Human Microbiome.

To explore a view of the human microbiome as an interconnected, functional, dynamic system that may be linked to the pathogenesis and progression of glaucoma. A literature review was undertaken that included publications from 1966 to 2023. Bacterial lipopolysaccharides (LPS) activate toll-like receptors (TLR) and mediate the human immune response. The LPS-TLR4 pathway is a potential avenue for the ocular, gut, and oral microbiomes to interface and/or influence ocular disease. Studies of gut dysbiosis have shown that alterations in the healthy microbiota can predispose the host to immune-mediated inflammatory and neurodegenerative conditions, while oral and ocular surface dysbiosis has been correlated with glaucoma. While developmental exposure to commensal microflora has shown to be necessary for the autoimmune and neurodegenerative responses to elevated intraocular pressure to take place, commensal bacterial products like short-chain fatty acids have regulatory effects protective against glaucoma. Alterations to human microbiotas have been associated with changes in intestinal permeability, gene regulation, immune cell differentiation, and neural functioning, which may predispose the host to glaucoma. Select microbes have been highlighted for their potential contributions to glaucoma disease progression or protection, raising the potential for microbiota-based treatment modalities. Current topical glaucoma treatments may disrupt the ocular surface microbiota, potentially having ramifications on host health. Further study of the relationships between human microbiome and glaucoma is needed.

POU6F2, a risk factor for glaucoma, myopia and dyslexia, labels specific populations of retinal ganglion cells

Pou6f2 is a genetic connection between central corneal thickness (CCT) in the mouse and a risk factor for developing primary open-angle glaucoma. POU6F2 is also a risk factor for several conditions in humans, including glaucoma, myopia, and dyslexia. Recent findings demonstrate that POU6F2-positive retinal ganglion cells (RGCs) comprise a number of RGC subtypes in the mouse, some of which also co-stain for Cdh6 and Hoxd10. These POU6F2-positive RGCs appear to be novel of ON–OFF directionally selective ganglion cells (ooDSGCs) that do not co-stain with CART or SATB2 (typical ooDSGCs markers). These POU6F2-positive cells are sensitive to damage caused by elevated intraocular pressure. In the DBA/2J mouse glaucoma model, heavily-labeled POU6F2 RGCs decrease by 73% at 8 months of age compared to only 22% loss of total RGCs (labeled with RBPMS). Additionally, Pou6f2−/− mice suffer a significant loss of acuity and spatial contrast sensitivity along with an 11.4% loss of total RGCs. In the rhesus macaque retina, POU6F2 labels the large parasol ganglion cells that form the magnocellular (M) pathway. The association of POU6F2 with the M-pathway may reveal in part its role in human glaucoma, myopia, and dyslexia.

Microbial Dynamics in Ophthalmic Health: Exploring the Interplay between Human Microbiota and Glaucoma Pathogenesis.

The human microbiome has a crucial role in the homeostasis and health of the host. These microorganisms along with their genes are involved in various processes, among these are neurological signaling, the maturation of the immune system, and the inhibition of opportunistic pathogens. In this sense, it has been shown that a healthy ocular microbiota acts as a barrier against the entry of pathogens, contributing to the prevention of infections. In recent years, a relationship has been suggested between microbiota dysbiosis and the development of neurodegenerative diseases. In patients with glaucoma, it has been observed that the microbiota of the ocular surface, intraocular cavity, oral cavity, stomach, and gut differ from those observed in healthy patients, which may suggest a role in pathology development, although the evidence remains limited. The mechanisms involved in the relationship of the human microbiome and this neurodegenerative disease remain largely unknown. For this reason, the present review aims to show a broad overview of the influence of the structure and composition of the human oral and gut microbiota and relate its dysbiosis to neurodegenerative diseases, especially glaucoma.

A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.

In a novel autoimmune and high-pressure glaucoma model a complex immune response is induced.

The neurodegenerative processes leading to glaucoma are complex. In addition to elevated intraocular pressure (IOP), an involvement of immunological mechanisms is most likely. In the new multifactorial glaucoma model, a combination of high IOP and optic nerve antigen (ONA) immunization leads to an enhanced loss of retinal ganglion cells accompanied by a higher number of microglia/macrophages in the inner retina. Here, we aimed to evaluate the immune response in this new model, especially the complement activation and the number of T-cells, for the first time. Further, the microglia/macrophage response was examined in more detail. Six-week-old wildtype (WT+ONA) and βB1-connective tissue growth factor high-pressure mice (CTGF+ONA) were immunized with 1 mg ONA. A wildtype control (WT) and a CTGF group (CTGF) received NaCl instead. Six weeks after immunization, retinae from all four groups were processed for immunohistology, RT-qPCR, and flow cytometry, while serum was used for microarray analyses. We noticed elevated numbers of C1q+ cells (classical complement pathway) in CTGF and CTGF+ONA retinae as well as an upregulation of C1qa, C1qb, and C1qc mRNA levels in these groups. While the complement C3 was only increased in CTGF and CTGF+ONA retinae, enhanced numbers of the terminal membrane attack complex were noted in all three glaucoma groups. Flow cytometry and RT-qPCR analyses revealed an enhancement of different microglia/macrophages markers, including CD11b, especially in CTGF and CTGF+ONA retinae. Interestingly, increased retinal mRNA as well as serum levels of the tumor necrosis factor α were found throughout the different glaucoma groups. Lastly, more T-cells could be observed in the ganglion cell layer of the new CTGF+ONA model. These results emphasize an involvement of the complement system, microglia/macrophages, and T-cells in glaucomatous disease. Moreover, in the new multifactorial glaucoma model, increased IOP in combination with autoimmune processes seem to enforce an additional T-cell response, leading to a more persistent pathology. Hence, this new model mimics the pathomechanisms occurring in human glaucoma more accurately and could therefore be a helpful tool to find new therapeutic approaches for patients in the future.

An Autotaxin-Induced Ocular Hypertension Mouse Model Reflecting Physiological Aqueous Biomarker.

Animal models of ocular hypertension (OH) have been developed to understand the pathogenesis of glaucoma and facilitate drug discovery. However, many of these models are fraught with issues, including severe intraocular inflammation and technical challenges. Lysophosphatidic acid (LPA) is implicated in trabecular meshwork fibrosis and increased resistance of aqueous outflow, factors that contribute to high intraocular pressure (IOP) in human open-angle glaucoma. We aimed to elevate IOP by increasing expression of the LPA-producing enzyme autotaxin (ATX) in mouse eyes. Tamoxifen-inducible ATX transgenic mice were developed. Tamoxifen was administered to six- to eight-week-old mice via eye drops to achieve ATX overexpression in the eye. IOP and retinal thickness were measured over time, and retinal flat-mount were evaluated to count retinal ganglion cells (RGCs) loss after three months. Persistent elevation of ATX expression in mouse eyes was confirmed through immunohistochemistry and LysoPLD activity measurement. ATX Tg mice exhibited significantly increased IOP for nearly two months following tamoxifen treatment, with no anterior segment changes or inflammation. Immunohistochemical analysis revealed enhanced expression of extracellular matrix near the angle after two weeks and three months of ATX induction. This correlated with reduced outflow facility, indicating that sustained ATX overexpression induces angle fibrosis, elevating IOP. Although inner retinal layer thickness remained stable, peripheral retina showed a notable reduction in RGC cell count. These findings confirm the successful creation of an open-angle OH mouse model, in which ATX expression in the eye prompts fibrosis near the angle and maintains elevated IOP over extended periods.

Microglial involvement in ocular hypertension

Glaucoma, a chronic and multifactorial neurodegenerative disease, is the leading cause of irreversible blindness. It is characterized by the degeneration of retinal ganglion cells (RGCs), loss of their axons, and damage to the lamina cribrosa. To study glaucomatous optic neuropathy, experimental rodent models are commonly used due to their low cost, ease of handling, and similarities to the human retina. The ideal model must reproduce tissue changes observed in human glaucoma, such as alterations of the optic nerve head, thinning of the retinal nerve fibre layer, activation of glial cells, and progressive degeneration of RGCs.The DBA/2J transgenic mouse model is the most commonly used genetic model, characterized by a gradual increase in intraocular pressure (IOP) with age, leading to rapid and progressive loss of RGCs and optic nerve degeneration. However, limitations such as iris atrophy and peripheral anterior synechiae can alter the anterior segment and influence IOP measurement. Induced models of ocular hypertension (OHT) have a predictable IOP increase following experimental procedures, but this increase is usually rapid and sustained for a relatively short time, which may not represent the slow and sustained IOP elevation observed in primary open‐angle glaucoma. Nonetheless, induced models produce similar pathophysiological mechanisms to genetic models. Other models include the injection of hypertonic saline into episcleral veins to cause scarring of the trabecular meshwork and elevate IOP, and the intracameral injection of polystyrene or magnetic microbeads to obstruct normal drainage flow of aqueous humour, resulting in an IOP increase. A new model employs an intracameral injection of a cross‐linking polymer that produces a viscosity increase, impeding normal drainage of the aqueous humour and resulting in an IOP increase and loss of RGCs.In this SIS, we review the different glaucoma models, their methods, and the retinal tissue changes, with a focus on microglial cell changes.

Nicotinamide Prevents Retinal Vascular Dropout in a Rat Model of Ocular Hypertension and Supports Ocular Blood Supply in Glaucoma Patients.

To investigate whether nicotinamide (NAM) modulates retinal vasculature in glaucoma. This was a prospective controlled clinical trial investigating animal and human histopathology. Participants included normotensive and ocular hypertensive rats, postmortem human ocular tissue, glaucoma patients (n = 90), and healthy controls (n = 30). The study utilized histopathology, computer-assisted retinal vasculature analysis, optical coherence tomography angiography (OCTA), and NAM treatment. The main outcome measures included retinal vascular parameters in rats as assessed by AngioTool; retinal vasculature integrity in rats and humans as assessed by histopathology, antibody-staining, and ImageJ-based measurements; and retinal perfusion density (PD) and flux index in humans as assessed by OCTA. A number of vessel parameters were altered in ocular hypertension/glaucoma compared to healthy controls. NAM treatment improved the retinal vasculature in ocular hypertensive rats, with an increase in mean vessel area, percentage area covered by vessels, total vessel length, total junctions, and junction density as assessed by AngioTool (all P < 0.05); vessel wall integrity as assessed by VE-cadherin antibody staining was also improved (P < 0.01). In humans, as assessed by OCTA, increases in PD in the optic nerve head and macula complete image (0.7%, P = 0.04 and 1.0%, P = 0.002, respectively) in healthy controls, and an increase in the temporal quadrant of the macula (0.7%, P = 0.02) in glaucoma patients was seen after NAM treatment. NAM can prevent retinal vascular damage in an animal model of glaucoma. After NAM treatment, glaucoma patients and healthy controls demonstrated a small increase in retinal vessel parameters as assessed by OCTA.