Laser-induced Mouse Research Articles

Hydrophobic vehicles for hydrophilic drugs: Sustained intravitreal caffeine delivery with oleogels.

Caffeine is the most widely consumed bioactive ingredient in the world, which has been found to show great therapeutic potential in several posterior eye diseases. While intravitreal injection represents the ideal administration route for these disorders, it remains challenging to achieve sustained release of caffeine in the vitreous cavity. Herein, we address this issue by loading crystalline caffeine within oleogels (Ca@oleogels), oily delivery vehicles which provide a hydrophobic environment that is opposite to the hydrophilic nature of their cargos. Mathematical modeling of the in vitro release profiles indicated the diffusion process of the drug from Ca@oleogels was playing a dominating role in caffeine release. Furthermore, sustained intravitreal delivery was evidenced by higher drug levels from 12 h until the end of the pharmacokinetic study (240 h) and a 3.2-fold reduction in Cmax compared to pristine caffeine. Superior therapeutic effects were obtained with oleogels in a laser-induced mouse choroidal neovascularization model. Advantages of Ca@oleogels as caffeine delivery vehicles included excellent biocompatibility, low cost and simplicity of manufacturing as well, which indicated they can be administrated safely and were readily amenable to scale-up production cost-effectively. Moreover, sustained release of another hydrophilic model drug (congo red) was also demonstrated with the same formulation design. Therefore, this strategy serves as a general solution to sustained intravitreal delivery of hydrophilic small molecule drugs.

CircSIRT2/miR-542-3p/VASH1 axis regulates endothelial-to-mesenchymal transition (EndMT) in subretinal fibrosis in age-related macular degeneration models.

Neovascular age-related macular degeneration (nAMD), characterized by choroidal neovascularization (CNV), is one of the leading causes of severe visual impairment and irreversible vision loss around the world. Subretinal fibrosis (SRF) contributes to the incomplete response to anti-vascular endothelial growth factor (VEGF) treatment and is one of the main reasons for long-term poor visual outcomes in nAMD. Reducing SRF is urgently needed in the anti-VEGF era. The role of non-coding RNAs has been implicated in CNV; however, their roles in SRF have not been elucidated yet. Herein, we comprehensively investigated circular RNA (circRNA) profiles in the laser-induced mouse SRF model and the transforming growth factor-β (TGF-β) induced human umbilical vein endothelial cell (HUVEC) fibrosis model. A novel circRNA, circSIRT2, was identified, and its function in SRF and endothelial-to-mesenchymal transition (EndMT) regulation was investigated. circSIRT2 was consistently upregulated in fibrotic models invivo and invitro. circSIRT2 overexpression downregulated the fibrotic markers and inhibited the proliferation and migration of endothelial cells invitro. circSIRT2 overexpression invivo also reduced SRF area in mice. Mechanistically, circSIRT2 functioned by sponging miR-542-3p, which further upregulated the expression of vasohibin-1 (VASH1) and reduced SRF lesion development. Vitreous delivery of miR-542-3p and VASH1 in the mouse SRF model also confirmed the pro-fibrotic function of miR-542-3p and anti-fibrotic function of VASH1, respectively. In conclusion, circSIRT2 inhibited SRF by binding miR-542-3p, which stimulated the VASH1 expression and subsequently suppressed EndMT. The circSIRT2/miR-542-3p/VASH1 axis may serve as a promising therapeutic target for SRF in nAMD.

An mRNA-encoded dominant-negative inhibitor of transcription factor RUNX1 suppresses vitreoretinal disease in experimental models.

Messenger RNA (mRNA)-based therapies are a promising approach to medical treatment. Except for infectious diseases, no other disease has mRNA-based therapies available. The eye is an ideal model for mRNA therapeutic development because it requires limited dosing. Proliferative vitreoretinopathy (PVR) is a blinding condition caused by retinal detachment that now lacks available medical treatment, with surgery as the only treatment option. We previously implicated runt-related transcription factor-1 (RUNX1) as a driver of epithelial-to-mesenchymal transition (EMT) in PVR and as a critical mediator of aberrant ocular angiogenesis when up-regulated. On the basis of these findings, an mRNA was designed to express a dominant-negative inhibitor of RUNX1 (RUNX1-Trap). We show that RUNX1-Trap delivered in polymer-lipidoid complexes or lipid nanoparticles sequestered RUNX1 in the cytosol and strongly reduced proliferation in primary cell cultures established from fibrotic membranes derived from patients with PVR. We assessed the preclinical efficacy of intraocular delivery of mRNA-encoded RUNX1-Trap in a rabbit model of PVR and in a laser-induced mouse model of aberrant angiogenesis often used to study wet age-related macular degeneration. mRNA-encoded RUNX1-Trap suppressed ocular pathology, measured as pathological scores in the rabbit PVR model and leakage and lesion size in the laser-induced choroidal neovascularization mouse model. mRNA-encoded RUNX1-Trap also strongly reduced proliferation in a human ex vivo explant model of PVR. These data demonstrate the therapeutic potential of mRNA-encoded therapeutic molecules with dominant-negative properties, highlighting the potential of mRNA-based therapies beyond standard gene supplementation approaches.

Segmentation and quantitative analysis of optical coherence tomography (OCT) images of laser burned skin based on deep learning

Evaluation of skin recovery is an important step in the treatment of burns. However, conventional methods only observe the surface of the skin and cannot quantify the injury volume. Optical coherence tomography (OCT) is a non-invasive, non-contact, real-time technique. Swept source OCT uses near infrared light and analyzes the intensity of light echo at different depths to generate images from optical interference signals. To quantify the dynamic recovery of skin burns over time, laser induced skin burns in mice were evaluated using deep learning of Swept source OCT images. A laser-induced mouse skin thermal injury model was established in thirty Kunming mice, and OCT images of normal and burned areas of mouse skin were acquired at day 0, day 1, day 3, day 7, and day 14 after laser irradiation. This resulted in 7000 normal and 1400 burn B-scan images which were divided into training, validation, and test sets at 8:1.5:0.5 ratio for the normal data and 8:1:1 for the burn data. Normal images were manually annotated, and the deep learning U-Net model (verified with PSPNe and HRNet models) was used to segment the skin into three layers: the dermal epidermal layer, subcutaneous fat layer, and muscle layer. For the burn images, the models were trained to segment just the damaged area. Three-dimensional reconstruction technology was then used to reconstruct the damaged tissue and calculate the damaged tissue volume. The average IoU value and f-score of the normal tissue layer U-Net segmentation model were 0.876 and 0.934 respectively. The IoU value of the burn area segmentation model reached 0.907 and f-score value reached 0.951. Compared with manual labeling, the U-Net model was faster with higher accuracy for skin stratification. OCT and U-Net segmentation can provide rapid and accurate analysis of tissue changes and clinical guidance in the treatment of burns.

Novel CCR3-targeted cyclic peptides as potential therapeutic agents for age-related macular degeneration via inhibiting angiogenesis and reducing retinal photoreceptor damage

Novel CCR3-targeted cyclic peptides as potential therapeutic agents for age-related macular degeneration via inhibiting angiogenesis and reducing retinal photoreceptor damage

PolySialic Acid Nanoparticles Actuate Complement-Factor-H-Mediated Inhibition of the Alternative Complement Pathway: A Safer Potential Therapy for Age-Related Macular Degeneration.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

Fibronectin binds integrin α5β1 to regulate macular neovascularization through the Wnt/β-catenin signaling pathway

Age-related macular degeneration (AMD) is a progressive, degenerative disease of the macula. The formation of macular neovascularization (MNV) and subretinal fibrosis of AMD is the most classic cause of the loss of vision in older adults worldwide. While the underlying causes of MNV and subretinal fibrosis remain elusive, the common feature of many common retinal diseases is changes the proportions of protein deposition in extracellular matrix (ECM) when compared to normal tissue. In ECM, fibronectin (FN) is a crucial component and plays a pivotal part not only in fibrotic diseases but also in the process of angiogenesis. The study aims to understand the role of ligand FN and its common integrin receptor α5β1 on MNV, and to understand the molecular mechanism involved. To study this, the laser-induced MNV mouse model and the rhesus macaque choroid-retinal endothelial cell line (RF/6A) chemical hypoxia mode were established, and the FN-α5β1 expression levels were detected by immunohistochemistry (IHC) and quantitative real-time PCR analysis (qRT-PCR). Fibronectin expression was silenced using small interfering RNA (siRNA) targeting FN. The tube formation and vitro scratch assays were used to assess the ability to form blood vessels and cell migration. To measure the formation of MNV, immunofluorescence, and Western blot assays were used. These results revealed that the expressions of FN and integrin α5β1 were distinctly increased in the laser-induced MNV mouse model and in the RF/6A cytochemically induced hypoxia model, and the expression tendency was identical. After the use of FN siRNA, the tube formation and migration abilities of the RF/6A cells were lower, the ability of endothelial cells to proliferate was confined and the scope of damage caused by the laser in animal models was significantly cut down. In addition, FN gene knockdown dramatically inhibited the expression of Wnt/β-catenin signal. The interaction of FN with the integrin receptor α5β1 in the constructed model, which may act through the Wnt/β-catenin signaling pathway, was confirmed in this study. In conclusion, FN may be a potential new molecular target for the prevention and treatment of subretinal fibrosis and MNV.

Emerging Co-Assembled and Sustained Released Natural Medicinal Nanoparticles for Multitarget Therapy of Choroidal Neovascularization.

Age-related macular degeneration (AMD) disease has become a worldwide senile disease, and frequent intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) is the mainstream treatment in the clinic, which is associated with sight-threatening complications. Herein, nintedanib, an inhibitor of angiogenesis, and lutein, a potent antioxidant, can co-assemble into nanoparticles through multiple noncovalent interactions. Interestingly, the co-assembled lutein/nintedanib nanoparticles (L/N NPs) exhibit significantly improved stability and achieve long-term sustained release of two drugs for at least two months in mice. Interestingly, in rabbit eyeball with a more complete barrier system, the L/N NPs still successfully distribute in the retina and choroid for a month. In the laser-induced mouse choroidal neovascularization model, the L/N NPs after a minimally invasive subconjunctival administration can successfully inhibit angiogenesis and achieve comparable and even better therapeutic results to that of standard intravitreal injection of anti-VEGF. Therefore, the subconjunctival injection of L/N NPs with long-term sustained drug release behavior represents a promising and innovative strategy for AMD treatment. Such minimally invasive administration together with the ability to effectively inhibit angiogenesis reduce inflammation and counteract oxidative stress and holds great potential for improving patient outcomes and quality of life in those suffering from this debilitating eye condition.

Neutrophil extracellular traps boost laser-induced mouse choroidal neovascularization through the activation of the choroidal endothelial cell TLR4/HIF-1α pathway.

Choroidal neovascularization (CNV) is characterized by the infiltration of immune cells, particularly neutrophils. Neutrophil extracellular trap (NET) facilitates the angiogenesis of pulmonary endothelial cells via activating Toll-like receptor 4 (TLR4). TLR4 promotes the expression of transcription factor hypoxia inducible factor-1α (HIF-1α), which promotes inflammation and angiogenesis via the up-regulation of metalloproteinase-9 (MMP-9) and interleukin-1β (IL-1β). In the present study, we aimed to identify the formation of NET and its role in CNV. Our results showed that NET levels were increased in a mouse laser-induced CNV model via oxidative stress, whereas the inhibition of NET alleviated CNV. In vitro, NET activated the TLR4/HIF-1α pathway in human choroidal endothelial cells (HCECs). Additionally, NET increased the transcription and expression of MMP-9 and IL-1β in HCECs via activating the TLR4/HIF-1α pathway. Meanwhile, NET promoted the inflammatory response accompanied by the proliferation, migration and tube formation of HCECs in a MMP-9- and IL-1β-dependent manner. In conclusion, NET was up-regulated in CNV and promoted the formation of CNV via activating the TLR4/HIF-1α pathway in choroidal endothelial cells. Our data uncovered the novel role of NET in promoting the formation of CNV. The underlying mechanism of NET could be targeted to delay the process of CNV.

LncRNA MALAT1 knockdown inhibits the development of choroidal neovascularization

In the pathogenesis of age-related macular degeneration, long non-coding RNAs have become important regulators. This study aimed to investigate the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the progression of choroidal neovascularization (CNV) and the underlying mechanisms. The in vivo and in vitro model of CNV was established using laser-induced mouse CNV model and human choroidal vascular endothelial cells (HCVECs) exposed to hypoxia respectively. We explore the role of MALAT1 in the pathogenesis of CNV by using the small interference RNA both in vivo and in vitro. MALAT1 expression was found to be upregulated in the retinal pigment epithelial-choroidal complexes. MALAT1 knockdown inhibited CNV development and leakage in vivo and decreased HCVECs proliferation, migration, and tube formation in vitro. MALAT1 performed the task as a miR-17-5p sponge to regulate the expression of vascular endothelial growth factor A (VEGFA) and E26 transformation specific-1 (ETS1). This study provides a new perspective on the pathogenesis of CNV and suggests that the axis MALAT/miR-17-5p/VEGFA or ETS1 may be an effective therapeutic target for CNV.

Luteolin inhibits subretinal fibrosis and epithelial-mesenchymal transition in laser-induced mouse model via suppression of Smad2/3 and YAP signaling

Subretinal fibrosis (SF) accounts for vision loss in patients with neovascular age-related macular degeneration (nAMD) even treated with adequate intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) drugs. Currently, there is no treatment available to prevent or treat SF caused by nAMD. This study aims to investigate the potential effects of luteolin on SF and epithelial-mesenchymal transition (EMT) as well as the underlying molecular pathways both in vivo and in vitro. Seven-week-old male C57BL/6J mice were employed to establish laser-induced choroidal neovascularization (CNV) and SF. One day after the laser induction, luteolin was administered intravitreally. SF and CNV were assessed with the immunolabeling of collagen type I (collagen I) and isolectin B4 (IB4), respectively. RPE65 and α-SMA colocalization in the lesions was used to evaluate the extent of EMT in retinal pigment epithelial (RPE) cells by using immunofluorescence. In vitro, luteolin was administered to TGFβ1-treated primary human RPE (phRPE) cells. RT-qPCR, Western blot and immunofluorescence were employed to evaluate the change of EMT-related molecules, epithelial markers, and relevant signaling pathways. The functional changes associated with EMT were investigated using the scratch assay, Transwell migration assay, and collagen gel contraction assay. CCK-8 was used to determine the cell viability of phRPE cells. On day 7 and 14 after laser induction in mice, intravitreal injection of luteolin dramatically decreased the immunolabeled sizes of both collagen I and IB4, as well as the amount of colocalized double immunostaining of α-SMA and RPE65 in laser-induced SF lesions. In vitro, TGFβ1-treated phRPE cells demonstrated increased cell migration and contraction capacity, accompanied with considerable overexpression of fibronectin, α-SMA, N-cadherin and vimentin, as well as downregulation of E-cadherin and ZO-1. The above changes were largely inhibited by luteolin co-incubation. Mechanistically, luteolin could evidently decrease the phosphorylation of Smad2/3, whereas increase the phosphorylation of YAP in TGFβ1-treated phRPE cells. This study demonstrates that luteolin exhibits the anti-fibrotic effect in a laser-induced mouse model by inhibiting EMT of RPE cells via deactivating Smad2/3 and YAP signaling, which provides a potential natural compound for the prevention and treatment of SF and fibrosis-related diseases.

Inhibition of AIF-1 alleviates laser-induced macular neovascularization by inhibiting endothelial cell proliferation via restrained p44/42 MAPK signaling pathway

Age-related macular degeneration (AMD) is a leading blinding disease worldwide, and macular neovascularization (MNV) is a common complication encountered in the advanced stages of AMD. While the underlying causes of MNV remain elusive, aberrant multiplication of choroidal endothelial cells (CECs) and increased vascular endothelial growth factor (VEGF) are thought to play significant roles in the occurrence and development of MNV. Allograft inflammatory factor-1(AIF-1) is a crucial regulatory factor of vascular tubular structure formation and growth, involving the proliferation and migration of vascular endothelial cells and various tumor cells. This study aimed to understand how AIF-1 effects the proliferation of CECs and the subsequent progression of MNV. To study this, a mouse MNV model was established through laser injury, and the AIF-1 expression levels were then measured using western blot and immunohistochemistry. AIF-1 siRNA was intravitreally injected to silence AIF-1 gene expression. Western blot and choroidal flat mount were performed to measure the progression of MNV and proliferation of the CECs. These results showed that the protein expression of AIF-1 was significantly elevated in the laser-induced mouse MNV model, and the expression trend was consistent with VEGF. The protein level of AIF-1 was significantly decreased after the intravitreal injection of AIF-1 siRNA, the damage range of laser lesions was significantly reduced, and the proliferation of endothelial cells was inhibited. Knockdown of the AIF-1 gene significantly inhibited the expression of mitogen-activated protein kinase p44/42 in MNV lesions. In summary, this research demonstrates that AIF-1 promoted MNV progression by promoting the proliferation of CECs and that silencing AIF-1 significantly ameliorates MNV progression in mouse models, which may act through the p44/42 MAPK signaling pathway. AIF-1 could be a new potential molecular target for MNV.

A laser-induced mouse model of progressive retinal degeneration with central sparing displays features of parafoveal geographic atrophy

There are no disease-modifying treatments available for geographic atrophy (GA), the advanced form of dry age-related macular degeneration. Current murine models fail to fully recapitulate the features of GA and thus hinder drug discovery. Here we describe a novel mouse model of retinal degeneration with hallmark features of GA. We used an 810 nm laser to create a retinal lesion with central sparing (RLCS), simulating parafoveal atrophy observed in patients with progressive GA. Laser-induced RLCS resulted in progressive GA-like pathology with the development of a confluent atrophic lesion. We demonstrate significant changes to the retinal structure and thickness in the central unaffected retina over a 24-week post-laser period, confirmed by longitudinal optical coherence tomography scans. We further show characteristic features of progressive GA, including a gradual reduction in the thickness of the central, unaffected retina and of total retinal thickness. Histological changes observed in the RLCS correspond to GA pathology, which includes the collapse of the outer nuclear layer, increased numbers of GFAP + , CD11b + and FcγRI + cells, and damage to cone and rod photoreceptors. We demonstrate a laser-induced mouse model of parafoveal GA progression, starting at 2 weeks post-laser and reaching confluence at 24 weeks post-laser. This 24-week time-frame in which GA pathology develops, provides an extended window of opportunity for proof-of-concept evaluation of drugs targeting GA. This time period is an added advantage compared to several existing models of geographic atrophy.

RO4929097, a Selective γ-Secretase Inhibitor, Inhibits Subretinal Fibrosis Via Suppressing Notch and ERK1/2 Signaling in Laser-Induced Mouse Model.

PurposeThis study aimed to explore whether RO4929097 (RO), a specific γ-secretase inhibitor, could inhibit the subretinal fibrosis in laser-induced mouse model and the relevant molecular mechanisms.MethodsMale C57BL/6J mice were used to produce choroidal neovascularization (CNV) and subretinal fibrosis by laser photocoagulation, and RO was administered intravitreally 1 day after laser induction. The sizes of CNV and subretinal fibrosis were measured and quantified in both 2D and 3D constructions. The ARPE-19 cell line and primary human RPE (phRPE) cells were treated with TGFβ1, in combination with or without RO, to examine Notch related molecules, epithelial mesenchymal transition (EMT), cell viability, migration, and contractile function, as well as the crosstalk between Notch and other EMT relevant signaling pathways.ResultsIntravitreal injection of RO reduced the sizes of both CNV and subretinal fibrosis in laser-induced young and old mice at day 7 and day 14 after laser induction. Moreover, EMT and Notch activation in RPE-choroid complexes from laser-induced mice were significantly attenuated by RO. In vitro, TGFβ1 activated Notch signaling and induced EMT in ARPE-19 cells, accompanied by enhanced EMT-related function, which were inhibited by RO. The inhibition of RO on EMT was further confirmed in TGFβ1-treated phRPE cells. Blockage of Notch signaling by RO could inhibit ERK1/2 signaling; whereas ERK1/2 inhibition had no effect on Notch. The action of RO was independent of Smad2/3 or p38, and co-inhibition of Notch and Smad2/3 showed synergistic effect on EMT inhibition.ConclusionsRO exerts its antifibrotic effect by directly inhibiting Notch signaling and indirectly suppressing ERK1/2 signaling. Targeting Notch signaling might provide a therapeutic strategy in prevention and treatment of subretinal fibrosis in neovascular age-related macular degeneration (nAMD).

Cytochrome P450 oxidase 2J inhibition suppresses choroidal neovascularization in mice

Choroidal neovascularization (CNV) in age-related macular degeneration (AMD) leads to blindness. It has been widely reported that increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) diets reduce CNV. Of the three major pathways metabolizing ω-3 (and ω-6 LCPUFA), the cyclooxygenase and lipoxygenase pathways generally produce pro-angiogenic metabolites from ω-6 LCPUFA and anti-angiogenic ones from ω-3 LCPUFA. Howevehr, cytochrome P450 oxidase (CPY) 2C produces pro-angiogenic metabolites from both ω-6 and ω-3 LCPUFA. The effects of CYP2J2 products on ocular neovascularization are still unknown. Understanding how each metabolic pathway affects the protective effect of ω-3 LCPUFA on retinal neovascularization may lead to therapeutic interventions. To investigate the effects of LCPUFA metabolites through CYP2J2 pathway and CYP2J2 regulation on CNV both in vivo and ex vivo. The impact of CYP2J2 overexpression and inhibition on neovascularization in the laser-induced CNV mouse model was assessed. The plasma levels of CYP2J2 metabolites were measured by liquid chromatography and tandem mass spectroscopy. The choroidal explant sprouting assay was used to investigate the effects of CYP2J2 inhibition and specific LCPUFA CYP2J2 metabolites on angiogenesis ex vivo. CNV was exacerbated in Tie2-Cre CYP2J2-overexpressing mice and was associated with increased levels of plasma docosahexaenoic acids. Inhibiting CYP2J2 activity with flunarizine decreased CNV in both ω-6 and ω-3 LCPUFA-fed wild-type mice. In Tie2-Cre CYP2J2-overexpressing mice, flunarizine suppressed CNV by 33 % and 36 % in ω-6, ω-3 LCPUFA diets, respectively, and reduced plasma levels of CYP2J2 metabolites. The pro-angiogenic role of CYP2J2 was corroborated in the choroidal explant sprouting assay. Flunarizine attenuated ex vivo choroidal sprouting, and 19,20-EDP, a ω-3 LCPUFA CYP2J2 metabolite, increased sprouting. The combined inhibition of CYP2J2 with flunarizine and CYP2C8 with montelukast further enhanced CNV suppression via tumor necrosis factor-α suppression. CYP2J2 inhibition augmented the inhibitory effect of ω-3 LCPUFA on CNV. Flunarizine suppressed pathological choroidal angiogenesis, and co-treatment with montelukast inhibiting CYP2C8 further enhanced the effect. CYP2 inhibition might be a viable approach to suppress CNV in AMD.

The Anti-Inflammatory Effect of Hydrogen Gas Inhalation and Its Influence on Laser-Induced Choroidal Neovascularization in a Mouse Model of Neovascular Age-Related Macular Degeneration.

Background: Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. Choroidal neovascularization (CNV) is the major pathologic feature of neovascular AMD. Oxidative damages and the ensuing chronic inflammation are representative of trigger events. Hydrogen gas (H2) has been demonstrated as an antioxidant and plays a role in the regulation of oxidative stress and inflammation. This experiment aimed to investigate the influence of H2 inhalation on a mouse model of CNV. Methods: Laser was used to induce CNV formation. C57BL/6J mice were divided into five groups: the control group; the laser-only group; and the 2 h, 5 h, and 2.5 h/2.5 h groups that received laser and H2 inhalation (21% oxygen, 42% hydrogen, and 37% nitrogen mixture) for 2 h, 5 h, and 2.5 h twice every day, respectively. Results: The severity of CNV leakage on fluorescence angiography showed a significant decrease in the H2 inhalation groups. The mRNA expression of hypoxia-inducible factor 1 alpha and its immediate downstream target vascular endothelial growth factor (VEGF) showed significant elevation after laser, and this elevation was suppressed in the H2 inhalation groups in an inhalation period length-related manner. The mRNA expression of cytokines, including tumor necrosis factor alpha and interlukin-6, also represented similar results. Conclusion: H2 inhalation could alleviate CNV leakage in a laser-induced mouse CNV model, and the potential mechanism might be related to the suppression of the inflammatory process and VEGF-driven CNV formation.

Gnotobiotic Operations and Assembly for Development of Germ-Free Animal Model of Laser-Induced Choroidal Neovascularization.

PurposeCompelling new evidence reveals a close link between the gut microbiome and the pathogenesis of neovascular age-related macular degeneration (nAMD). Germ-free (GF) animal models are the current gold standard for studying host the microbe interactions in vivo; yet, no GF animal models of nAMD are available today. This protocol describes gnotobiotic operations and assembly for a laser-induced choroidal neovascularization (CNV) model in GF mice to study the gut microbiome in neovascular AMD.MethodsWe developed a step-wise approach to performing retinal laser photocoagulation in GF C57BL/6J mice that were bred and maintained at the gnotobiotic facility. Following a strict sterility protocol, we administered laser photocoagulation via an Argon 532-nm laser attached to a customized slit-lamp delivery system. Sterility was confirmed by weekly fecal cultures and reverse transcriptase–polymerase chain reaction.ResultsThe experiment was repeated twice at different time points using seven mice (14 eyes). Stool cultures and RT-PCR remained negative for 14 days post-procedure in all mice. Lectin immunostaining performed on choroidal flatmounts confirmed the presence of CNV lesions 2 weeks after laser treatment.ConclusionsWe established a GF mouse model of nAMD with detailed guidelines to deliver retinal laser in GF mice maintaining sterility after the laser procedure.Translational RelevanceTo our knowledge, this is the first protocol that describes a GF murine model of laser-induced CNV. In addition to nAMD, this animal model can be used to investigate host–microbial interactions in other eye diseases with laser-induced mouse models such as glaucoma and retinal vein occlusion.

Effect of Retinal Injury Induced by Laser Photocoagulation on Visuospatial Memory in Mouse Model

Visual pathway reveals the connection between neuronal activity of the brain and eye. The neural networks of brain amplify the retinal signals resulting in the formation of visual image. The laser injury in the retina may affect the visual pathway and may lead to disruption of neuronal signals/activity. Therefore, we aimed to study the effect of retinal injury induced by laser on cognitive abilities in laser-induced mouse model. We have established laser model to understand the relation between retina and brain by disrupting retinal pigment epithelial (RPE) layer and evaluate the effect of laser-induced retinal injury on visuospatial memory. Age- and sex-matched C57BL/6J male mice were taken for conducting the experiments. The laser model was established by using laser photocoagulator to disrupt the RPE layer of the retina. After defined irradiation of laser onto mouse retina, the fundus fluorescein angiography was performed to confirm the laser spots. The visuospatial and short-term memory was performed using neurobehavioral test, that is, Morris water maze (MWM), and passive avoidance, respectively. In MWM experiment, results showed that escape latency time, which was taken by healthy and laser-injured mice was comparable. This was further validated by another neurobehavioral analysis, that is, passive avoidance that showed nonsignificant difference between these two groups using independent t -test. Visuospatial memory may not be affected by retinal injury induced by laser photocoagulation. It may depend on the power of the laser and duration of the laser. The severe injury in the retina such as optic nerve damage may cause dysfunctioning of visual pathway.

Echinomycin mitigates ocular angiogenesis by transcriptional inhibition of the hypoxia-inducible factor-1

Echinomycin (EKN), an inhibitor of hypoxia-inducible factor (HIF)-1 DNA-binding activity, has been implied as a possible therapeutic agent in ischemic diseases. Here, we assess EKN in hypoxia-driven responses in vitro using human primary adult retinal pigment epithelium cells (aRPE) and retinal endothelial cells (hREC), and in vivo using the laser-induced mouse choroidal neovascularization (CNV) model. Effects of EKN on hypoxia-mediated pathways in aRPE were analyzed by Western blotting for HIF-1α protein, quantitative PCR of HIF-target genes, and proteome array for soluble angiogenic factors. In vitro inhibition of angiogenesis by EKN was determined in hREC. In vivo inhibition of angiogenesis by EKN was determined in the mouse laser-induced CNV, as a model of HIF-associated ocular neovascularization. CNV lesion area was determined by fundus fluorescein angiography. aRPE treated with EKN showed hypoxia-dependent significantly decreased cell recovery in the wound healing assay. These results were supported by lower levels of HIF-mediated transcripts detected in hypoxic aRPE cells treated with EKN compared with non-treated controls, and confirmed by proteome profiler for angiogenic factors. hREC exposed to aRPE EKN-conditioned medium displayed reduced sprouting angiogenesis. Mice with laser-induced CNV treated with intravitreally injected EKN showed significantly decreased vascular lesion area when compared with a mouse equivalent of aflibercept, or vehicle-treated controls. Our data proposes EKN as a potent inhibitor of HIF-mediated angiogenesis in retinal cells and in the mouse model of CNV, which could have future implications in the treatment of patients with neovascular age-related macular degeneration.

Time course of collateral vessel formation after retinal vein occlusion visualized by OCTA and elucidation of factors in their formation

BackgroundIt is clinically recognized that collateral vessels can form after retinal vein occlusion (RVO) in some cases and these vessels can lead to spontaneous recovery of the pathological condition. In recent years, optical coherence tomography angiography (OCTA) has become a decisive clinical instrument. Unlike previous angiography tests, OCTA enables the non-invasive visualization of fundus vasculature without the need for administration of a contrast agent. However, it remains to be determined if OCTA depicts the ‘true’ histological status as several studies have reported artifacts in OCTA imaging.MethodsWe generated a laser-induced mouse RVO model, and evaluated the subsequent formation of collateral vessels in order to understand the mechanisms by which collateral vessels form using OCTA imaging, as well as molecular and histological assessments.ResultsWe succeeded in visualizing the time course of collateral vessel formation in a mouse RVO model and confirmed the similarity in formation of collateral vessels only within the deep layer of the retina in both human and mouse. We hypothesized that sphingosine 1-phosphate receptor-1 (S1PR1) may play important roles via vascular shear stress linking vein occlusion and collateral vessel formation. Results from OCTA revealed that collateral vessels are increased in response to administration of a S1PR1 agonist in a mouse RVO model. Based on quantitative reverse transcription polymerase chain reaction (qRT-PCR), S1PR1 messenger ribonucleic acid (mRNA) levels in the whole retina peaked 6 h after photocoagulation in this model. Immunohistochemical staining of retinal flat mounts revealed that S1PR1 staining occurred along the laser-occluded blood vessels.ConclusionWe observed the temporal process of collateral vessel formation in a mouse RVO model and identified the relationship between S1PR1 and shear stress as one of the factors in collateral vessel formation in RVO.