Experimental eye research最新文献

Due to its unique physiological structure and functions, the eye has received considerable attention in the field of Adeno-associated virus (AAV) gene therapy. Inherited retinal degenerative diseases, which arise from pathogenic mutations in mRNA transcripts expressed in the eye's photoreceptor cells or retinal pigment epithelium (RPE), are the most common cause of vision loss. However, current retinal gene therapy mostly involves subretinal injection of therapeutic genes, which treats a limited area, entails retinal detachment, and requires sophisticated techniques. Intravitreal (IVT) injection provides an alternative method with less invasion and more convenience for retinal gene therapy. In the present study, we performed a directed evolution via DNA shuffling in RHO-GFP mice and identified a novel recombinant AAV vector (AAV-M04) suitable for IVT injection in the gene delivery of retinal tissue. Compared with AAV2, AAV9, and AAV2.7m8, AAV-M04 vector exhibited higher transduction efficiency in retinal ganglion cell line-5 (RGC-5) cells as well as in human embryonic stem cell derived retinal organoids. Importantly, when delivered via IVT injection in mice, the AAV-M04 vector also showed better delivery efficiency of transgene as indicated by the red fluorescence protein mScarlet. The red fluorescence was distributed in a wider retinal area of AAV-M04 injected mice, suggesting the potent retinal targeting of AAV-M04 vector. In addition, AAV-M04 qualities including the packaging efficiency, the thermal stability, and the capsid integrity were superior to controls, which were important in drug manufacture. In summary, we screened a novel AAV-M04 vector with great retinal-targeting via IVT injection, which provides the potential of AAV-M04 for effective gene therapy of retinal diseases.

The P2X7 receptor (P2X7R) for extracellular ATP is implicated in several forms of retinal degeneration, including diabetic retinopathy, age-related macular degeneration, and glaucoma. P2X7R stimulation can trigger release of master cytokine IL-1β from microglia in the brain and from macrophages, but evidence of release from retinal microglia is indirect. Isolated mouse and rat retinal microglia, and wholemounts from CX3CR1^+/GFP mice, were examined to determine if ATP induced IL-1β release directly from retinal microglial cells and if it also primed expression of IL-1β on an mRNA and protein level. Isolated retinal microglia were ramified and expressed low levels of polarization markers unless provoked. Over 90% of isolated microglial cells expressed P2X7R, with cytoplasmic Ca²⁺ elevation following receptor stimulation. ATP induced a dose-dependent release of IL-1β from primed microglial cells that was blocked by P2X7R antagonist A839977 and emulated by agonist BzATP. P2X7R stimulation also primed Il1b mRNA in isolated microglia cells. BzATP increased IL-1β immunostaining and GFP fluorescence throughout lamina of retinal wholemounts from CX3CR1^+/GFP mice. Some of the IL-1β and GFP signals colocalized, particularly in the outer retina, and in projections extending distally through photoreceptor layers. The inner retina had more microglia without IL-1β, and more IL-1β staining without microglia. Substantial IL-1β release was also detected from rat retinal microglial cells, but not optic nerve head astrocytes. In summary, this study implicates microglial cells as a key source of released IL-1β when levels of extracellular ATP are increased following retinal damage, and suggest a greater participation in the outer retina.

The abrupt and substantial elevation of intraocular pressure (IOP) in acute glaucoma induces retinal ischemia/reperfusion (I/R) injury, resulting in progressive retinal ganglion cell (RGC) death and irreversible visual impairment. PANoptosis, a form of regulated cell death consisting of pyroptosis, apoptosis and necroptosis, is reported to be involved in high IOP-induced RGC death. However, the precise mechanisms of RGC death remain unclear, and neuroinflammation is considered to play a vital role. TAT-N24, a synthetic inhibitor targeting the p55 regulatory subunit of phosphatidylinositol 3-kinase (p55PIK) signaling, demonstrates anti-inflammatory effect in uveitis and may have certain neuroprotective effects. Therefore, we investigated whether TAT-N24 could shield RGCs from immunoinflammatory damage in an acute glaucoma mouse model and explored the potential mechanism associated with PANoptosis. A mouse model of acute ocular hypertension (AOH) was established. Intravitreal injection of TAT-N24 was conducted to evaluate its impact on RGC death. The expression levels of key components in PANoptosis were analyzed using RT-qPCR and Western blotting. Immunohistochemistry and immunofluorescence staining on eyeball sections were employed to assess the expression of p55PIK, Brn3a, and ionized calcium binding adaptor molecule 1 (Iba1). Retinal structure was examined by H&E staining, while cell apoptosis was evaluated by TdT-mediated dUTP nick end labeling (TUNEL). The results showed that intravitreal injection of TAT-N24 effectively alleviated RGC death and retinal damage induced by AOH injury. The key components in PANoptosis were markedly upregulated after AOH injury, while these components were significantly inhibited after TAT-N24 treatment. Moreover, the expression levels of Z-DNA-binding protein 1 (ZBP1)-PANoptosome (ZBP1, RIPK1, RIPK3, and Caspase-8), NLR family pyrin domain-containing protein 3 (NLRP3), and NLR family CARD domain-containing protein 4 (NLRC4) inflammasomes were notably elevated after AOH injury, which was significantly suppressed by TAT-N24. In conclusion, PANoptosis was involved in AOH-induced RGC death and retinal damage. TAT-N24 exhibited an anti-PANoptotic effect, protecting RGCs by inhibiting ZBP1-PANoptosome as well as NLRP3 and NLRC4 inflammasomes after AOH injury.

Subretinal injection of gene products is the only treatment option for inherited retinal diseases. However, this procedure induces localized high multiaxial deformations, potentially causing irreversible tissue damage due to retinal overstretching and tearing. Comprehensive characterization of retinal mechanical behavior is essential for performing safe injections. Although uniaxial tensile test has been used in the literature, it has many limitations for retinal characterization. To date, retinal mechanical properties are poorly understood due to the lack of standardized testing protocol. This study aimed to introduce a combined experimental-computational approach using small punch testing and finite element simulations to investigate retina elastic behavior under biaxial deformations. To develop a suitable testing protocol for retinal samples, we evaluated the impact of environmental conditions on retinal elasticity by performing uniaxial tensile tests on porcine retinal strips in air, in a saline bath, and at different temperatures. The results showed that conditions did not significantly affect the elastic modulus. We then developed an easy and reproducible small punch test protocol, allowing to measure for the first time the load-displacement response of the retina under biaxial deformations. Computational simulations enabled the analysis of retinal deformations and the identification of its elastic modulus (5.5 kPa). The outcomes of this study highlight the great potential of the combined approach as a viable alternative to uniaxial tensile test to advance the understanding of retinal biomechanics. This is essential not only for minimizing sight-threatening surgical complications during injections, but also for building predictive in silico models, and developing biomimetic scaffolds.

Diabetic retinopathy is a major ocular complication associated with diabetes mellitus. Pericyte loss is a hallmark of diabetic retinopathy. The platelet-derived growth factor (PDGF)-B-PDGF receptor-β (PDGFRβ) signaling pathway plays an important role in the proliferation and migration of pericytes. Imatinib, an antineoplastic drug primarily used to treat chronic myelogenous leukemia, inhibits the PDGFRβ tyrosine kinase. In this study, we aimed to determine the time-course of pathological changes in the retinal vasculature following pharmacological depletion of pericytes with imatinib. Rats were injected with imatinib once daily for 1, 2, or 4 days starting on postnatal day (P) 4. The distribution of endothelial cells and pericytes in the retina was assessed at P4, P5, P6, P8, and P11. Single and multiple injections of imatinib (100 mg/kg) significantly decreased the pericyte coverage within the retinal capillaries on the day after the completion of each injection protocol. After pericyte coverage decreased, endothelial cell degeneration and microaneurysm formation were initiated. Following the elimination of the inhibitory effect of imatinib on the PDGFRβ signaling pathway, the pericyte coverage returned to control levels but structural abnormalities of the retinal vasculature with microaneurysms and dense capillaries were observed. Vascular pathological features are similar to those of the early clinical manifestations of diabetic retinopathy. Therefore, these rats could serve as animal models to study the mechanisms underlying the pathological changes that occur after pericyte loss in diabetic retinopathy.

The mechanisms underlying the low incidence of myopia at high altitudes remain unclear. Choroidal thickness and the dopaminergic system have been shown to be closely associated with myopia development. This study aimed to investigate the effects of high altitude exposure on choroidal thickness and the dopaminergic system. Mice were subjected to acute hypobaric hypoxia at an altitude of 5000 m for durations ranging from 2 to 72 h, as well as chronic exposure at an altitude of 3670 m for a period of 3 months. Choroidal thickness was assessed using hematoxylin and eosin (H&E) staining of ocular tissues. The retinal dopamine (DA) levels and its primary metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were quantified via high-performance liquid chromatography (HPLC). The expression levels of dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R) were evaluated using immunofluorescence techniques. Study results indicated that choroidal thickness significantly increased after 6 h of high altitude exposure. Retinal dopamine levels showed significant increases in both the 2-10 h and 3 months high altitude groups. Conversely, retinal DOPAC levels decreased in the 2 h and 4 h groups but increased significantly at 72 h. Following high altitude exposure, D1R expression correlated positively with DA levels, while D2R expression exhibited a negative correlation. In conclusion, high-altitude exposure is associated with significant increases in choroidal thickness and retinal DA levels, with D1R and D2R expression patterns varying in response to changes in retinal DA. These findings may represent a key molecular mechanism contributing to the lower incidence of myopia observed at high altitudes.

Animal models that help us understand how elevated intraocular pressure (IOP) causes axonal injury will lead to new glaucoma therapies. Because reliable measurements are difficult to obtain in chronic models, we developed the controlled elevation of IOP (CEI) approach. Here, a cannula connected to an elevated balanced salt solution (BSS) reservoir is inserted into the anterior chamber of anesthetized Brown Norway rats. The extent and duration of IOP exposure is controlled by adjusting the reservoir height. We now describe a method for creating CEI in awake animals. A Pinport, which has a silicone plug that can be penetrated repeatedly, is modified, attached to the skull, and connected to a microcannula that is implanted in the posterior chamber. To elevate IOP, BSS from a reservoir is allowed to flow through a pressure transducer to a swivel-mounted tether and injector. The injector is placed on the Pinport, bypassing the need for anterior chamber cannulation and general anesthesia during CEI. The surgical technique and equipment required for implantation are described, as well as the equipment and methods for performing awake CEI in several animals at a time. The ability of this system to control the level of IOP is demonstrated by TonoLab measurement, and by comparing reservoir (Pinport) pressures to direct measurement using an independent anterior chamber cannula and transducer. We also demonstrate that IOP elevation can be maintained over several hours. Specific pitfalls during and after surgical implantation are highlighted to help other researchers adopt these techniques.

Autophagy is common in the aging retinal pigment epithelium (RPE). A dysfunctional autophagy in aged RPE is implicated in the pathogenesis of age-related macular degeneration. Aging human retina accompanies degenerative changes in photoreceptor mitochondria. It is not known how the damaged mitochondria are handled by photoreceptor cells with aging. This study examined donor human retinas (age: 56-94 years; N = 12) by transmission electron microscopy to find mitochondrial dynamics and status of autophagy in macular photoreceptor cells. Observations were compared between the relatively lower age (56-78 years) and aged retinas (80-94 years). Mitochondrial fusion was predominant in photoreceptor inner segments (ellipsoids), but rarely seen in the synaptic terminals. Also, fusion became widespread with progressive aging in ellipsoids (12% and 21% between rods and cones at tenth decade, respectively). More importantly, it was found that the photoreceptor synaptic mitochondria altered significantly with aging (swelling and loss of cristae), compared to those in ellipsoids that became dark and condensed. The damaged synaptic mitochondria were sequestered inside autophagosomes, whose frequency was higher in aged photoreceptors, being 34% in cone and 24% in rod terminals, at tenth decade. However, autolysosomes/residual bodies were rare, and thus the aged photoreceptor synaptic terminals harboured many autophagosomes, the possible reasons for which are discussed. Such age-related altered mitochondrial population and defective autophagy in synaptic terminals may influence photoreceptor survival in late aging.

Retinopathy of prematurity (ROP) is a proliferative retinal vascular disorder that critically affects the visual development of premature infants, potentially leading to irreversible vision loss or even blindness. Despite its significance, the underlying mechanisms of this disease remain insufficiently understood. In this study, we utilized the oxygen-induced retinopathy (OIR) mouse model and conducted endothelial functional assays to explore the role of Sterol Regulatory Element-Binding Protein 1 (SREBF1) in ROP pathogenesis. SREBF1 expression levels, along with its downstream targets, were investigated through Western blotting, RT-qPCR, and immunofluorescence staining techniques. Furthermore, Co-Immunoprecipitation (Co-IP) was employed to examine the molecular mechanisms involved. Our results demonstrated a significant increase in SREBF1 expression in both the OIR mouse model and hypoxic primary human retinal microvascular endothelial cells (HRMECs). Interventions conducted both in vivo and in vitro showed notable efficacy in reducing pathological neovascularization. Importantly, we discovered that SREBF1 plays a key role in modulating lipid metabolism in HRMECs by regulating the expression of ACC1 and FASN, leading to cellular reprogramming. This reprogramming influences HRMEC proliferation, migration, and tube formation through the HIF-1α/TGF-β signaling pathway, ultimately contributing to pathological retinal neovascularization. These findings provide new insights into the role of SREBF1 in angiogenesis within the context of ROP, offering potential therapeutic targets for the management and treatment of this disease.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease linked to aging. This study investigates potential connections between IPF and age-related eye problems using a bleomycin-induced IPF mouse model. Intratracheal administration of bleomycin induces rapid lung injury in mice, followed by IPF with characteristics of cellular senescence. IPF-injured mice had reduced amplitudes of scotopic ERG and immunostaining of visual arrestin, suggesting declined rod-related visual function. Interestingly, the mice's eyes also showed increased susceptibility to Staphylococcus aureus infections, reminiscent of the aging eyes. To determine whether an early onset of aging contributes to the eye disorders, we examined complement and senescence markers in the retina. In bleomycin-injury IPF mice, DNA damage-related senescence marker γH2AX was found in the retinal out nuclear layer where photoreceptors are located. Additionally, IPF mice displayed elevated levels of C3b, a complement fragment resulting from C3 activation that occurs frequently in aging eyes. These findings underscore the potential of IPF as a valuable mouse model for investigating early-onset age-related ocular disorders.