Single-cell transcriptomic Atlas of aging macaque ocular outflow tissues.

The progressive degradation in the trabecular meshwork (TM) is related to age-related ocular diseases like primary open-angle glaucoma. However, the molecular basis and biological significance of the aging process in TM have not been fully elucidated. Here, we established a dynamic single-cell transcriptomic landscape of aged macaque TM, wherein we classified the outflow tissue into 12 cell subtypes and identified mitochondrial dysfunction as a prominent feature of TM aging. Furthermore, we divided TM cells into 13 clusters and performed an in- depth analysis on cluster 0, which had the highest aging score and the most significant changes in cell proportions between the two groups. Ultimately, we found that the APOE gene was an important differentially expressed gene in cluster 0 during the aging process, highlighting the close relationship between cell migration and extracellular matrix regulation, and TM function. Our work further demonstrated that silencing the APOE gene could increase migration and reduce apoptosis by releasing the inhibition on the PI3K-AKT pathway and downregulating the expression of extracellular matrix components, thereby increasing the aqueous outflow rate and maintaining intraocular pressure within the normal range. Our work provides valuable insights for future clinical diagnosis and treatment of glaucoma.

Serine to proline mutation at position 341 of MYOC impairs trabecular meshwork function by causing autophagy deregulation.

Glaucoma is a highly heritable disease, and myocilin was the first identified causal and most common pathogenic gene in glaucoma. Serine-to-proline mutation at position 341 of myocilin (MYOCS341P) is associated with severe glaucoma phenotypes in a five-generation primary open-angle glaucoma family. However, the underlying mechanisms are underexplored. Herein, we established the MYOCS341P transgenic mouse model and characterized the glaucoma phenotypes. Further, we systematically explored the functional differences between wild-type and MYOCS341P through immunoprecipitation, mass spectrometry, and RNA-seq analyses. We found that MYOCS341P transgenic mice exhibit glaucoma phenotypes, characterized by reduced aqueous humor outflow, elevated intraocular pressure, decreased trabecular meshwork (TM) cell number, narrowed Schlemm's canal, retinal ganglion cell loss, and visual impairment. Mechanistically, the secretion of dysfunctional MYOCS341P accumulated in the endoplasmic reticulum (ER), inducing ER stress and dysregulation of autophagy, thereby promoting TM cell death. We describe an effective transgenic model for mechanistic studies and the screening of therapeutic targets. Our data generated from high-throughput analyses help elucidate the mechanism underlying mutant MYOC-related glaucoma.

Early Proteomic Characteristics and Changes in the Optic Nerve Head, Optic Nerve, and Retina in a Rat Model of Ocular Hypertension.

The pathogenesis of glaucoma is still unknown. There are few studies on the dynamic change of tissue-specific and time-specific molecular pathophysiology caused by ocular hypertension (OHT). This study aimed to identify the early proteomic alterations in the retina, optic nerve head (ONH), and optic nerve (ON). After establishing a rat model of OHT, we harvested the tissues from control and glaucomatous eyes and analyzed the changes in protein expression using a multiplexed quantitative proteomics approach (TMT-MS3). Our study identified 6403 proteins after 1-day OHT and 4399 proteins after 7-days OHT in the retina, 5493 proteins after 1-day OHT and 4544 proteins after 7-days OHT in ONH, and 5455 proteins after 1-day OHT and 3835 proteins after 7-days OHT in the ON. Of these, 560 and 489 differential proteins were identified on day 1 and 7 after OHT in the retina, 428 and 761 differential proteins were identified on day 1 and 7 after OHT in the ONH, and 257 and 205 differential proteins on days 1 and 7 after OHT in the ON. Computational analysis on day 1 and 7 of OHT revealed that alpha-2 macroglobulin was upregulated across two time points and three tissues stably. The differentially expressed proteins between day 1 and 7 after OHT in the retina, ONH, and ON were associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, oxidative stress, microtubule, and crystallin. And the most significant change in retina are crystallins. We validated this proteomic result with the Western blot of crystallin proteins and found that upregulated on day 1 but recovered on day 7 after OHT, which are promising as therapeutic targets. These findings provide insights into the time- and region-order mechanisms that are specifically affected in the retina, ONH, and ON in response to elevated IOP during the early stages.

Cerebrospinal Fluid Pressure Reduction Induces Glia-Mediated Retinal Inflammation and Leads to Retinal Ganglion Cell Injury in Rats.

Low intracranial pressure (LICP)-induced translaminar cribrosa pressure difference (TLCPD) elevation has been proven as a risk factor in glaucomatous neurodegeneration, whereas the underlying retinal immune features of LICP-induced retinal ganglion cells (RGC) injury remain elusive. Here, we identified the retinal immune characteristics of LICP rats, and minocycline (Mino) treatment was utilized to analyze its inhibitory role in glia-mediated retinal inflammation of LICP rats. The results showed that retrograde axonal transport was decreased in LICP rats without significant RGC loss, indicating the RGC injury was at an early stage before the morphological loss. The activation of retinal microglia and astrocytes with morphologic and M1 or A1-marker alternations was detected in TLCPD elevation rats, the activation level is more dramatic in HIOP rats than in the LICP rats (P<0.05). Besides, we detected reduced retinal tight junction protein expressions, accompanied by specific imbalance patterns of T lymphocytes in the retina of both LICP and HIOP rats (P<0.05). Further Mino treatment showed an effective inhibitory role in glia-driven inflammatory responses in LICP rats, including improving retrograde axonal transport, inhibiting retinal glial activation and proinflammatory subtype polarization, and alleviating the blood-retina barrier compromise. This study identified the glia-mediated retinal inflammation features triggered by LICP stimulus, and Mino application exhibited an effective role in the inhibition of retinal glia-mediated inflammation in LICP-induced TLCPD elevation rats.

Biomechanical homeostasis in ocular diseases: A mini-review.

Diabetes mellitus-induced hyperglycemia is responsible for multiple pathological ocular alternations from vasculopathy to biomechanical dyshomeostasis. Biomechanical homeostasis is crucial to maintain the normal physiological condition of the eyes. Biomechanical features vary in eye tissues regarding different anatomical positions, tissue components, and cellular functions. The disturbance in biomechanical homeostasis may result in different ocular diseases. In this review, we provide a preliminary sketch of the latest evidence on the mechano-environment of the eyeball and its possible influencing factors, thereby underscoring the relationship between the dyshomeostasis of ocular biomechanics and common eye diseases (e.g., diabetic retinopathy, keratoconus, glaucoma, spaceflight-associated neuro-ocular syndrome, retinal vein occlusion and myopia, etc.). Together with the reported evidence, we further discuss and postulate the potential role of biomechanical homeostasis in ophthalmic pathology. Some latest strategies to investigate the biomechanical properties in ocular diseases help unveil the pathological changes at multiple scales, offering references for making new diagnostic and treatment strategies targeting mechanobiology.

Reference values for trans-laminar cribrosa pressure difference and its association with systemic biometric factors.

OBJECTIVES: To provide reference values of trans-laminar cribrosa pressure difference (TLCPD) and reveal the association of TLCPD with systemic biometric factors. METHODS: In this cross-sectional study, 526 quasi-healthy subjects (including 776 eyes) who required lumbar puncture for medical reasons were selected from 4915 neurology inpatients from 2019 to 2022. Patients with any diseases affecting intraocular pressure (IOP) or intracranial pressure (ICP) were excluded. The ICPs of all subjects were obtained by lumbar puncture in the left lateral decubitus position. IOP was measured in the seated position by a handheld iCare tonometer prior to lumbar puncture. TLCPD was calculated by subtracting ICP from IOP. Systemic biometric factors were assessed within 1 h prior to TLCPD measurement. RESULTS: The TLCPD (mean ± standard deviation) was 4.4 ± 3.6 mmHg, and the 95% reference interval (defined as the 2.5th-97.5th percentiles) of TLCPD was -2.27 to 11.94 mmHg. The 95% reference intervals for IOP and ICP were 10-21 and 6.25-15.44 mmHg, respectively. IOP was correlated with ICP (r = 0.126, p < 0.001). TLCPD was significantly negatively correlated with body mass index (r = -0.086, p = 0.049), whereas it was not associated with age, gender, height, weight, blood pressure, pulse, or waist and hip circumference. CONCLUSIONS: This study provides reference values of TLCPD and establishes clinically applicable reference intervals for normal TLCPD. Based on association analysis, TLCPD is higher in people with lower BMI.

Impact of PTEN/SOCS3 deletion on amelioration of dendritic shrinkage of retinal ganglion cells after optic nerve injury.

Retinal ganglion cell (RGC) degeneration, leading to irreversible blindness in chronic optic neuropathies, commonly begins with dendritic shrinkage followed by axon degeneration. Although limited axon regeneration in the optic nerve is possible with a genetic deletion of PTEN/SOCS3 after optic nerve injury, the roles of PTEN/SOCS3 on dendritic preservation and regeneration remain unclear. This study investigated the effect of PTEN/SOCS3 genetic deletion on the structural integrity of RGC dendrites and axons in the retina following optic nerve crush. Using time-lapse, in vivo confocal scanning laser ophthalmoscopy to serially image dendritic and axonal arborizations of RGCs over six months after injury, RGC dendrites and axons were only preserved in Thy-1-YFP/PTEN-/- and Thy-1-YFP/PTEN-/-SOCS3-/- mice, and axons in the retina regenerated at a rate of 21.1 µm/day and 15.5 µm/day, respectively. By contrast, dendritic complexity significantly decreased in Thy-1-YFP-SOCS3-/- and control mice at a rate of 7.0 %/day and 7.1 %/day, respectively, and no axon regeneration in the retina was observed. RGC survival probability was higher in Thy-1-YFP/PTEN-/- and Thy-1-YFP/PTEN-/-SOCS3-/- mice compared with Thy-1-YFP-SOCS3-/- and control mice. The differential responses between the transgenic mice demonstrate that although a genetic deletion of PTEN, SOCS3, or PTEN/SOCS3 allows partial axon regeneration in the optic nerve after optic nerve crush, a deletion of PTEN, but not SOCS3, ameliorates RGC dendritic shrinkage. This shows that the signaling pathways involved in promoting axon regeneration do not equally contribute to the preservation of dendrites, which is crucial to the translational application of neuroregenerative therapies for visual restoration.

Intracameral injection of a chemically cross-linked hydrogel to study chronic neurodegeneration in glaucoma.

Investigation of neurodegeneration in glaucoma, a leading cause of irreversible blindness worldwide, has been obfuscated by the lack of an efficient model that provides chronic, mild to moderate elevation of intraocular pressure (IOP) with preservation of optical media clarity for long term, in vivo interrogation of the structural and functional integrity of the retinal ganglion cells (RGCs). Here, we designed and formulated an injectable hydrogel based on in situ cross-linking of hyaluronic acid functionalized with vinyl sulfone (HA-VS) and thiol groups (HA-SH). Intracameral injection of HA-VS and HA-SH in C57BL/6J mice exhibited mild to moderate elevation of IOP with daily mean IOP ranged between 14 ±â€¯3 and 24 ±â€¯3 mmHg, which led to progressive, regional loss of RGCs evaluated with in vivo, time-lapse confocal scanning laser ophthalmoscopy; a reduction in fractional anisotropy in the optic nerve and the optic tract projected from the eye with increased IOP in diffusion tensor magnetic resonance imaging; a decrease in positive scotopic threshold response in electroretinography; and a decline in visual acuity measured with an optokinetic virtual reality system. The proportion of RGC loss was positively associated with the age of the animals, and the levels and the duration of IOP elevation. The new glaucoma model recapitulates key characteristics of human glaucoma which is pertinent to the development and pre-clinical testing of neuroprotective and neuroregenerative therapies. STATEMENT OF SIGNIFICANCE: A new model to study chronic neurodegeneration in glaucoma has been developed via intracameral injection of a specifically designed hyaluronic acid functionalized with vinyl sulfone and thiol groups for cross-linking. Intracameral injection of the chemically cross-linked hydrogel generates mild to moderate IOP elevation, resulting in progressive degeneration of the retinal ganglion cells, optic nerve, and optic tract, and a decline in visual function. The model recapitulates the key features of neurodegeneration in human glaucoma, which will facilitate and expedite the development of neuroprotective and neuroregenerative therapies.