Developing a synergistic rate-retarding polymeric implant for controlling monoclonal antibody delivery in minimally invasive glaucoma surgery.

Monoclonal antibodies (mAbs) have garnered substantial attention within the field of ophthalmology and can be used to suppress scar formation after minimally invasive glaucoma surgeries. Here, by controlling mAb passive diffusion, we developed a polymeric, rate-controlling membrane reservoir loaded with poly(lactic-co-glycolic acid) microspheres to deliver mAb for several weeks. Different parameters were tested to ensure that the microspheres achieved a good quality characteristic, and our results showed that 1 %W/V emulsifier with 5 %W/V NaCl achieved mAb-loaded microspheres with the highest stability, encapsulation efficiency and minimal burst release. Then, we fabricated and compared 10 types of microporous films based on polylactic acid (PLA), polycaprolactone (PCL), and polyethylene glycol (PEG). Our results revealed distinct pore characteristics and degradation patterns in different films due to varying polymer properties, and all the polymeric film formulations showed good biocompatibility in both human trabecular meshwork cells and human conjunctival fibroblasts. Finally, the optimized microspheres were loaded into the reservoir-type polymeric implant assembled by microporous membranes with different surface coating modifications. The implant formulation, which was fabricated by 60 PCL: 40 PEG (3 %W/V) polymer with 0.1 %W/V poly(lactic-co-glycolic acid) barrier, exerted the best drug release profile that can sustained release mAb (83.6 %) for 4 weeks.

Inhibition of TLR4/NF-κB pathway and endoplasmic reticulum stress by overexpressed S100A4 ameliorates retinal ischemia-reperfusion injury of mice.

Retinal ischemia exists in various ischemic retinopathies including glaucoma, contributing to the death of retinal neurons. Calcium binding protein S100A4 is important in tumors, and our previous study found that S100A4 protects retinal ganglion cells (RGCs) against retinal ischemia-reperfusion (I/R) injury. This study was aimed to further discuss the neuroprotection and mechanisms of S100A4 in retinal I/R of mice. The rAAV-EF1α-s100a4-EGFP-WPRE or rAAV-EF1α-EGFP-WPRE-Pa was injected intravitreally 4 weeks before I/R. S100A4, molecules in TLR4 signaling pathway and endoplasmic reticulum (ER) stress branches, inflammatory molecules, and surviving RGCs and cholinergic amacrine (ChAT) cells were determined by quantitative PCR, western blot, or immunofluorescent staining. The apoptosis and necrosis of retinal neurons induced by I/R were inhibited by overexpressed S100A4. RGCs, ChAT cells, and the retinal function were preserved by S100A4 overexpressing 7 days after I/R. Mechanistically, the beneficial effects of S100A4 may be mediated by inhibiting the activation of TLR4 signaling pathway and alleviating ER stress, leading to the attenuation of inflammatory response of the retina after I/R. Our findings indicated that S100A4 has neuroprotective effect against retinal I/R injury, and promoting S100A4 expression may be an effective strategy to inhibit retinal neurons from degeneration in ischemic retinopathy.

COG1410 regulates microglial states and protects retinal ganglion cells in retinal ischemia-reperfusion injury.

Progressive loss of retinal ganglion cells (RGCs) caused by retinal ischemia-reperfusion (IR) injury can lead to irreversible vision impairment, with neuroinflammatory responses playing an important role in this process. COG1410, a mimetic peptide of apolipoprotein E, has demonstrated protective potential in the central nervous system, but its effects on retinal IR injury remain unexplored. In this study, we established a mouse model of retinal IR injury to investigate the effects of COG1410 on retinal microglia and RGCs. We observed CD16/32-marked and CD206-marked microglia and RGCs using immunofluorescence staining, detected the expression of inflammatory factors by PCR, and evaluated retinal apoptosis with TUNEL staining. We further investigated the potential mechanism by detecting the expression of key proteins via Western blot. The results reveal that COG1410 decreased the number of CD16/32-marked microglia and increased the number of CD206-marked microglia, alleviated the expression of IL-1ß and TNF-α, and reduced the loss of RGCs by inhibiting the mitochondrial-related apoptotic pathway. COG1410 was found to increase the expression of ERK1/2 and Nr4a1 but decrease the expression of NF-κB. The expression of TREM2 showed an increasing trend after COG1410 administration, but it was not statistically significant. In conclusion, COG1410 regulates microglial states and protects RGCs in retinal IR injury, showing promising potential for the treatment of eye diseases.

Adrenaline blocks key cell cycle genes and exhibits antifibrotic and vasoconstrictor effects in glaucoma surgery.

Adrenaline is a sympathomimetic drug used to maintain pupil dilation and to decrease the risk of bleeding. The aim of this study was to demonstrate if adrenaline could exert antifibrotic effects in glaucoma surgery. Adrenaline was tested in fibroblast-populated collagen contraction assays and there was a dose-response decrease in fibroblast contractility: matrices decreased to 47.4% (P = 0.0002) and 86.6% (P = 0.0036) with adrenaline 0.0005% and 0.01%, respectively. There was no significant decrease in cell viability even at high concentrations. Human Tenon's fibroblasts were also treated with adrenaline (0%, 0.0005%, 0.01%) for 24 h and RNA-Sequencing was performed on the Illumina NextSeq 2000. We carried out detailed gene ontology, pathway, disease and drug enrichment analyses. Adrenaline 0.01% upregulated 26 G1/S and 11 S-phase genes, and downregulated 23 G2 and 17 M-phase genes (P < 0.05). Adrenaline demonstrated similar pathway enrichment to mitosis and spindle checkpoint regulation. Adrenaline 0.05% was also injected subconjunctivally during trabeculectomy, PreserFlo Microshunt and Baerveldt 350 tube surgeries, and patients did not experience any adverse effects. Adrenaline is a safe and cheap antifibrotic drug that significantly blocks key cell cycle genes when used at high concentrations. Unless contraindicated, we recommend subconjunctival injections of adrenaline (0.05%) in all glaucoma bleb-forming surgeries.

3D-printed long-acting 5-fluorouracil implant to prevent conjunctival fibrosis in glaucoma.

OBJECTIVES: To develop a sustained release 5-fluorouracil (5-FU) implant by three-dimensional (3D) printing to effectively prevent conjunctival fibrosis after glaucoma surgery. METHODS: 3D-printed implants composed of polycaprolactone (PCL) and chitosan (CS) were fabricated by heat extrusion technology and loaded with 1% 5-FU. Light microscopy and scanning electron microscopy were used to study the surface morphology. The 5-FU concentration released over 8 weeks was measured by ultraviolet visible spectroscopy. The effects on cell viability, fibroblast contractility and the expression of key fibrotic genes were assessed in human conjunctival fibroblasts. KEY FINDINGS: The PCL-CS-5-FU implant sustainably released 5-FU over 8 weeks and the peak concentration was over 6.1 µg/ml during weeks 1 and 2. The implant had a smooth surface and its total weight decreased by 3.5% after 8 weeks. The PCL-CS-5-FU implant did not affect cell viability in conjunctival fibroblasts and sustainably suppressed fibroblast contractility and key fibrotic genes for 8 weeks. CONCLUSIONS: The PCL-CS-5-FU implant was biocompatible and degradable with a significant effect in suppressing fibroblast contractility. The PCL-CS-5-FU implant could be used as a sustained release drug implant, replacing the need for repeated 5-FU injections in clinic, to prevent conjunctival fibrosis after glaucoma surgery.

Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery.

The primary cause of failure for minimally invasive glaucoma surgery (MIGS) is fibrosis in the trabecular meshwork (TM) that regulates the outflow of aqueous humour, and no anti-fibrotic drug is available for intraocular use in MIGS. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a promising anti-fibrotic target. This study aims to utilise a novel lipid nanoparticle (LNP) to deliver MRTF-B siRNA into human TM cells and to compare its effects with those observed in human conjunctival fibroblasts (FF). Two LNP formulations were prepared with and without the targeting peptide cΥ, and with an siRNA concentration of 50 nM. We examined the biophysical properties and encapsulation efficiencies of the LNPs, and evaluated the effects of MRTF-B silencing on cell viability, key fibrotic genes expression and cell contractility. Both LNP formulations efficiently silenced MRTF-B gene and were non-cytotoxic in TM and FF cells. The presence of cΥ made the LNPs smaller and more cationic, but had no significant effect on encapsulation efficiency. Both TM and FF cells also showed significantly reduced contractibility after transfection with MRTF-B siRNA LNPs. In TM cells, LNPs with cΥ achieved a greater decrease in contractility compared to LNPs without cΥ. In conclusion, we demonstrate that the novel CL4H6-LNPs are able to safely and effectively deliver MRTF-B siRNA into human TM cells. LNPs can serve as a promising non-viral gene therapy to prevent fibrosis in MIGS.

Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage.

Purpose: Heat shock protein B8 (HspB8) can be upregulated rapidly in many pathologic processes, but its role in traumatic optic neuropathy remains unclear. In this study, we investigated the involvement of autophagy in the effects of HspB8 by using the optic nerve crush (ONC) model. Methods: Male C57BL/6J mice were intravitreally injected with recombinant adeno-associated virus type 2 (AAV2-shHspB8 or AAV2-GFP) and subsequently received ONC by a self-closing tweezers. Western blot and immunohistochemistry staining were used to evaluate the expression of HspB8. We conducted retinal flat-mount immunofluorescence to measure the quantities of retinal ganglion cells (RGCs), and full-field flash electroretinogram (ff-ERG) and optomotor response (OMR) were used to evaluate retinal function. The autophagy level was reflected by western blot, immunohistochemistry staining, and transmission electron microscope (TEM) images. We also applied 3-methyladenine (3MA) and rapamycin (Rapa) to regulate autophagy level in optic nerve injury. Results: ONC stimulated the expression of HspB8. Declines of RGCs and ff-ERG b-wave amplitudes resulting from ONC can be alleviated by HspB8 downregulation. Increased autophagy activity after ONC was observed; however, this change can be reversed by intravitreal injection of AAV2-shHspB8. Furthermore, application of autophagy inhibitor 3MA had the same neuroprotective effects as AAV2-shHspB8, as illustrated by ff-ERG and quantities of RGCs. Also, protection of AAV2-shHspB8 was compromised by the autophagy activator Rapa. Conclusions: Inhibition of HspB8 in mice optic nerve injury had neuroprotective effects, which may be derived from its downregulation of autophagy.

Krüppel-like factor 7 protects retinal ganglion cells and promotes functional preservation via activating the Akt pathway after retinal ischemia-reperfusion injury.

OBJECTIVE: The purpose of this study is to investigate the effects of Krüppel-like factor 7 (KLF7) on retinal ganglion cells (RGCs) and retinal function after retinal ischemia-reperfusion (RIR) injury in mice. METHODS: Male C57BL/6J mice were intravitreally injected with recombinant adeno-associated vectors (rAAV-KLF7-EGFP or rAAV-EGFP), and subsequently used to induce RIR injury. Retinal cryosections were used to access the efficacy of virus transfection, 1, 2, 3, and 4 weeks after rAAV-KLF7-EGFP transfer. RGCs survival rate was observed and quantified by immunofluorescent staining, 7 days after RIR injury. Meanwhile, electroretinogram (ERG) and optomotor response were used to evaluate the electrophysiological functions and visual acuity. Apoptosis was evaluated by TUNEL staining 1 day after RIR injury. Expression of KLF7, Akt, phospho-Akt, Bcl-2, and Bax were further detected by western blot to excavate the underlying mechanism. RESULTS: The transfection efficiency of rAAV-KLF7-EGFP was increased in a time-dependent manner, and the number of EGFP-positive cells was increased significantly 3 weeks after rAAV-KLF7-EGFP transfer. RGCs survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR, and visual acuity of mice were decreased after RIR injury. With the increase of light intensity, the amplitudes of scotopic ERG a- and b-wave were gradually increased while the incubation period was gradually shortened. RGCs survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR, and visual acuity of mice were increased after rAAV-KLF7-EGFP transfer. The protein level of KLF7 was up-regulated after rAAV-KLF7-EGFP transfer. Up-regulation of KLF7 significantly inhibited cells apoptosis, increased phospho-Akt and Bcl-2 expression, and decreased Bax expression. There were no significant changes in Akt expression. CONCLUSION: Overexpression of KLF7 can not only prevent the loss of RGCs, but also preserve the electrophysiological function. In addition, overexpression of KLF7 can ameliorate the retinal dysfunction after RIR injury, and ultimately improve the visual acuity of mice. The activation of Akt pathway and the suppression of the mitochondrial apoptotic pathway contribute to the neuroprotection of KLF7.

Prenatal Exposure to Retrorsine Induces Developmental Toxicity and Hepatotoxicity of Fetal Rats in a Sex-Dependent Manner: The Role of Pregnane X Receptor Activation.

Pyrrolizidine alkaloids (PAs) are a type of natural phytotoxin that contaminate food and feed and become an environmental health risk to humans and livestock. PAs exert toxicity that requires metabolic activation by cytochrome P450 (CYP) 3A, and case reports showed that fetuses are quite susceptible to PAs toxicity. The aim of this study was to explore the characteristics of developmental toxicity and fetal hepatotoxicity induced by retrorsine (RTS, a typcial toxic PA) and the underlying mechanism. Pregnant Wistar rats were intragastrically administered with 20 mg/(kg·day) RTS from gestation day (GD) 9 to 20. Results showed that prenatal RTS exposure lowered fetal bodyweights, reduced hepatocyte numbers, and potentiated hepatic apoptosis in fetuses, particularly females. Simutaneously, RTS increased CYP3A expression and pregnane X receptor (PXR) activation in female fetal liver. We further confirmed that RTS was a PXR agonist in LO2 and HepG2 cell lines. Furthermore, agonism or antagonism of androgen receptor (AR) either induced or blocked RTS-mediated PXR activation, respectively. As a PXR agonist, RTS toxicity was exacerbated in female fetus due to the increased CYP3A induction and self-metabolism, while the inhibitory effect of AR on PXR activation reduced the susceptibility of male fetus to RTS. Our findings indicated that PXR may be a potential therapeutic target for PA toxicity.

miR-7a Targets Insulin Receptor Substrate-2 Gene and Suppresses Viability and Invasion of Cells in Diabetic Retinopathy Mice via PI3K-Akt-VEGF Pathway.

INTRODUCTION: Diabetic retinopathy (DR) is one of the major leading causes for vision loss globally. Current study illustrates the role of miR-7a in DR. MATERIAL AND METHODS: Retinal pericytes (RPs) and Endothelial cells (ECs) were isolated from mouse model of DR. qRT-PCR was done for expression of miR-7a and target gene mRNA, Western blot for protein expression. Identification of miR-7a target gene was done by TargetScan and Luciferase assay. Cell viability and invasion was done by MTT and Transwell chamber assay. RESULTS: The expression of miR-7a was down-regulated whereas level of IRS-2 was unregulated in isolated RPs and ECs. Luciferase assay suggested correlation between miR-7a and IRS-2, over-expression of miR-7a using a mimic resulted in suppression in viability and invasion capacity of RPs and ECs and inhibited the protein levels of PI3K/Akt cascade and IRS-2, and however the inhibitor reversed them respectively. Transfection of siRNA targeting IRS-2 caused alteration in miR-7a mediated changes in ECs suggesting that miR-7a may decrease angiogenesis in DR by inhibiting the levels of IRS-2. CONCLUSION: miR-7a suppresses PI3K/Akt cascade via targeting IRS-2, thus decreasing the viability and invasion capacity of RPs and ECs, suggesting an interesting treatment target for DR.

Overexpression of S100A4 protects retinal ganglion cells against retinal ischemia-reperfusion injury in mice.

BACKGROUND: Glaucoma is characterized by the neurodegeneration of retinal ganglion cells (RGCs) and the optic nerve. Numerous studies have reported that S100A4 participates in the metastasis of tumor cells and nerve protection. This study was intended to explore the role of S100A4 on RGCs under retinal ischemia-reperfusion (I/R) injury in mice. METHODS: C57BL/6J mice were used to induce retinal I/R injury. The intravitreal administration of rAAV-EF1α-s100a4-EGFP-WPRE (rAAV-S100A4) or rAAV-EF1α-EGFP-WPRE-Pa was performed 4 weeks before I/R injury. Expression of S100A4 was detected by quantitative real-time PCR, immunofluorescence staining of retinal sections and western blot. Surviving RGCs were quantified using immunofluorescence staining. Staining of TUNEL was utilized to evaluate the apoptosis of retinal cells. Electroretinogram (ERG) was used to analyze retinal function. Expression of Akt, phospho-Akt, Bcl-2, and Bax were determined using western blotting to investigate the potential mechanisms of S100A4. RESULTS: Retinal S100A4 level had no statistical difference 7 days after I/R injury. The rAAV-S100A4 was clearly demonstrated by the green fluorescence protein in many layers of the retina after intravitreal injection and up-regulated the expression of S100A4. I/R injury resulted in an increase of the apoptosis of retinal cells and the reduction of surviving RGCs, however, overexpressed S100A4 inhibited the apoptosis of cells and a decrease of RGCs. ERG analysis showed a drop on amplitude of a-wave and b-wave was impeded to some extent by overexpressing of S100A4. Up-regulation of S100A4 raised the expression of phospho-Akt and reduced Bax expression. Nevertheless, there were no significant changes in the levels of Bcl-2 and total Akt. CONCLUSION: Our results indicate the neuroprotective effects of overexpressed S100A4 on RGCs by activating the Akt pathway and then inhibiting the apoptosis of cells after I/R injury. The use of S100A4 protein may be a novel therapeutic strategy for glaucoma.

SIRT1 is required for the neuroprotection of resveratrol on retinal ganglion cells after retinal ischemia-reperfusion injury in mice.

PURPOSE: Retinal ganglion cells (RGCs) loss is closely related to visual impairment in glaucoma, so the neuroprotection on RGCs is important and novel for glaucoma research. SIRT1, a family member of sirtuins, is implicated in many crucial processes of eye diseases. The purpose of this study is to determine the neuroprotection of SIRT1 on RGCs and to investigate the underlying mechanisms of these effects in an experimental model for acute glaucoma. METHODS: Retinal ischemia-reperfusion (IR) injury was induced in C57BL/6J mice. Resveratrol (RSV, activator of SIRT1) and sirtinol (inhibitor of SIRT1) were injected intravitreally 1 day before IR injury. RGCs survival rate was quantified by immunofluorescence staining. RGCs apoptosis was evaluated by the staining of TUNEL and cleaved caspase-3, and SIRT1 level was detected by western blot. Expressions of phospho-Akt, Akt, Bax, and Bcl-2 were further determined by western blot to investigate the neuroprotective mechanisms of SIRT1. RESULTS: RGCs survival rates and SIRT1 levels were decreased over time after IR injury. Intravitreal injection of RSV remarkably attenuated RGCs loss in a dose-dependent manner, and the most effective concentration of RSV was 100 µM. Up-regulation of SIRT1 by RSV significantly inhibited RGCs apoptosis, increased p-Akt level, decreased Bax and cleaved caspase-3 expressions, and all these effects were diminished by 100 µM sirtinol. Moreover, there were no significant changes in total Akt and Bcl-2 levels. CONCLUSION: SIRT1 activation by RSV confers neuroprotection on RGCs in retinal IR injury through the activation of Akt pathway and subsequent suppression of mitochondrial apoptotic pathway. Determination of the effective concentration of intravitreal injection of RSV also provides a theoretical basis for the clinical application of RSV.

Sex difference in monocrotaline-induced developmental toxicity and fetal hepatotoxicity in rats.

Pyrrolizidine alkaloids (PAs) are a class of hepatic toxins widely existing in plants. Cytochromes P450 (CYP) mediates PA bioactivation and toxicities in mammals. It has been reported that PAs can induce developmental toxicity, but systematic research is lacking. In this study, we investigated developmental toxicity of monocrotaline (MCT) in rats. Pregnant rats were administered with MCT (20 mg/kg) intragastrically from gestation day 9 to 20, followed by determination of changes in fetal growth, hepatic morphology, serum biochemical indices, and indicators of hepatocytes apoptosis. MCT was found to induce developmental toxicity and fetal hepatotoxicity, particularly in female fetuses. Metabolic activation was also studied by examination of bioactivation efficiency of MCT in fetal liver microsomes, serum MCT, pyrrole-protein adduction derived from MCT, and hepatic CYP3 A expression of fetuses in vivo. Male fetuses showed greater basal MCT bioactivation than that of female fetuses, but continuous exposure to MCT caused a selective CYP3 A induction in female fetuses, which may contribute to the sex difference in MCT-induced developmental toxicity.

Maternal-Fetal Disposition and Metabolism of Retrorsine in Pregnant Rats.

Pyrrolizidine alkaloids (PAs) are extensively synthesized by plants, are commonly present in herbs and foodstuffs, and exhibit hepatotoxicity requiring metabolic activation by cytochrome P450 3A to form the electrophilic metabolites-pyrrolic esters. PAs also cause embryo toxicity, but the metabolic profiles of PAs in fetus and placenta have been far from clear. In this study, we determined the basal metabolic activation of retrorsine (RTS) in rat maternal liver, placenta, and fetal liver in vitro and examined the fetal toxicity and bioactivation of RTS in vivo. Detection of microsomal RTS metabolites in vitro showed that the basal metabolic activity of fetal liver and placenta to RTS was much weaker than that of maternal liver. In addition, a higher rate of pyrrolic ester formation was found in normal male fetal liver compared with that of female pups. In vivo exposure to RTS caused fetal growth retardation, as well as placental and fetal liver injury. Little difference in serum RTS was observed in dams and fetuses, but the content of pyrrole-protein adduction in the fetal liver was much lower than that in maternal liver, which was consistent with basal metabolic activity. Unexpectedly, compared with basal metabolism in fetal liver, exposure to RTS during middle and late pregnancy caused an opposite gender difference in RTS metabolism and CYP3A expression in the fetal liver. For the first time, our study showed that RTS can permeate the placenta barrier and entering fetal circulation, whereas the intrauterine pyrrolic metabolite was generated mainly by fetal liver but not transported from the maternal circulation. Induction of CYP3A by RTS was gender-dependent in the fetal liver, which was probably responsible for RTS-induced fetal hepatic injury, especially for female pups.