Sui Wang, Postdoctoral Faculty Sponsor
A protocol to inject ocular drug implants into mouse eyes.
2022; 3 (1): 101143
Ocular drug implants (ODIs) are beneficial for treating ocular diseases. However, the lack of a robust injection approach for small-eyed model organisms has been a major technical limitation in developing ODIs. Here, we present a cost-effective, minimally invasive protocol to deliver ODIs into the mouse vitreous called Mouse Implant Intravitreal Injection (MI3). MI3 provides two alternative surgical approaches (air-pressure or plunger) to deliver micro-scaled ODIs into milli-scaled eyes, and expands the preclinical platforms to determine ODIs' efficacy, toxicity, and pharmacokinetics. For complete details on the use and execution of this protocol, please refer to Sun etal. (2021).
View details for DOI 10.1016/j.xpro.2022.101143
View details for PubMedID 35141566
Identification of cis-regulatory modules for adeno-associated virus-based cell type-specific targeting in the retina and brain.
The Journal of biological chemistry
Adeno Associated Viruses (AAVs) targeting specific cell types are powerful tools for studying distinct cell types in the central nervous system (CNS). Cis-regulatory modules (CRMs), e.g., enhancers, are highly cell type-specific and can be integrated into AAVs to render cell type specificity. Chromatin accessibility has been commonly used to nominate CRMs, which have then been incorporated into AAVs and tested for cell type-specificity in the CNS. However, chromatin accessibility data alone cannot accurately annotate active CRMs, as many chromatin-accessible CRMs are not active and fail to drive gene expression in vivo. Using available large-scale datasets on chromatin accessibility, such as those published by the ENCODE project, here we explored strategies to increase efficiency in identifying active CRMs for AAV-based cell type-specific labeling and manipulation. We found that pre-screening of chromatin-accessible putative CRMs based on the density of cell type-specific transcription factor binding sites (TFBSs) can significantly increase efficiency in identifying active CRMs. In addition, generation of synthetic CRMs by stitching chromatin-accessible regions flanking cell type-specific genes can render cell type-specificity in many cases. Using these straightforward strategies, we generated AAVs that can target the extensively studied interneuron and glial cell types in the retina and brain. Both strategies utilize available genomic datasets and can be employed to generate AAVs targeting specific cell types in CNS without conducting comprehensive screening and sequencing experiments, making a step forward in cell type-specific research.
View details for DOI 10.1016/j.jbc.2022.101674
View details for PubMedID 35148987
An intravitreal implant injection method for sustained drug delivery into mouse eyes.
Cell reports methods
2021; 1 (8)
Using small molecule drugs to treat eye diseases carries benefits of specificity, scalability, and transportability, but their efficacy is significantly limited by a fast intraocular clearance rate. Ocular drug implants (ODIs) present a compelling means for the slow and sustained release of small molecule drugs inside the eye. However, methods are needed to inject small molecule ODIs into animals with small eyes, such as mice, which are the primary genetic models for most human ocular diseases. Consequently, it has not been possible to fully investigate efficacy and ocular pharmacokinetics of ODIs. Here, we present a robust, cost-effective, and minimally invasive method called "mouse implant intravitreal injection" (MI3) to deliver ODIs into mouse eyes. This method will expand ODI research to cover the breadth of human eye diseases modeled in mice.
View details for DOI 10.1016/j.crmeth.2021.100125
View details for PubMedID 35128514
Identifcation of cis-regulatory elements that allow for AAV-based Muller glial-specific labeling and manipulation in the retina
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2021
View details for Web of Science ID 000690760500356
Cell type- and stage-specific expression of Otx2 is regulated by multiple transcription factors and cis-regulatory modules in the retina.
Development (Cambridge, England)
Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is necessary. We analyzed how the expression of the homeobox TF, Orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during development in the retina. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages. We conclude that retinal cells utilize a cohort of TFs with different expression patterns, and multiple CRMs with different chromatin configurations, to precisely regulate the expression of Otx2.
View details for DOI 10.1242/dev.187922
View details for PubMedID 34004867
Low-Luminance Blue Light-Enhanced Phototoxicity in A2E-Laden RPE Cell Cultures and Rats.
International journal of molecular sciences
2019; 20 (7)
N-retinylidene-N-retinylethanolamine (A2E) and other bisretinoids are components of lipofuscin and accumulate in retinal pigment epithelial (RPE) cells-these adducts are recognized in the pathogenesis of retinal degeneration. Further, blue light-emitting diode (LED) light (BLL)-induced retinal toxicity plays an important role in retinal degeneration. Here, we demonstrate that low-luminance BLL enhances phototoxicity in A2E-laden RPE cells and rats. RPE cells were subjected to synthetic A2E, and the effects of BLL on activation of apoptotic biomarkers were examined by measuring the levels of cleaved caspase-3. BLL modulates the protein expression of zonula-occludens 1 (ZO-1) and paracellular permeability in A2E-laden RPE cells. Early inflammatory and angiogenic genes were also screened after short-term BLL exposure. In this study, we developed a rat model for A2E treatment with or without BLL exposure for 21 days. BLL exposure caused fundus damage, decreased total retinal thickness, and caused neuron transduction injury in the retina, which were consistent with the in vitro data. We suggest that the synergistic effects of BLL and A2E accumulation in the retina increase the risk of retinal degeneration. These outcomes help elucidate the associations between BLL/A2E and angiogenic/apoptotic mechanisms, as well as furthering therapeutic strategies.
View details for DOI 10.3390/ijms20071799
View details for PubMedID 30979028
Novel Protective Effects of Cistanche Tubulosa Extract Against Low-Luminance Blue Light-Induced Degenerative Retinopathy.
Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
2018; 51 (1): 63-79
Blue light-emitting diode light (BLL)-induced phototoxicity plays an important role in ocular diseases and causes retinal degeneration and apoptosis in human retinal pigment epithelial (RPE) cells. Cistanche tubulosa extract (CTE) is a traditional Chinese medicine with many beneficial protective properties; however, few studies have examined the ocular protective roles of CTE. In this study, we investigated the mechanisms underlying the effects of CTE on BLL-induced apoptosis in vitro and in vivo.RPE cells were applied in the current in vitro study and cell viability was determined by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis-related protein expression was determined by western blot analysis and immunofluorescence staining. Brown Norway rats were used to examine exposure to commercially available BLL in vivo. Hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and western blot assays were used to examine retinal morphological deformation.CTE significantly inhibited hydrogen peroxide-, tert-butyl hydroperoxide-, sodium azide-, and BLL-induced RPE damage. Further, CTE reduced the expression of apoptotic markers such as cleaved caspase-3 and TUNEL staining after BLL exposure by inactivating apoptotic pathways, as shown via immunofluorescent staining. In addition, CTE inhibited the BLL-induced phosphorylation of c-Jun N-terminal kinase, extra signal-related kinases 1/2, and p38 in RPE cells. In vivo, the oral administration of CTE rescued 60-day periodic BLL exposure-induced decrements in retinal thickness and reduced the number of TUNEL-positive cells in the brown Norway rat model.CTE is a potential prophylactic agent against BLL-induced phototoxicity.
View details for DOI 10.1159/000495162
View details for PubMedID 30439705
Editor's Highlight: Periodic Exposure to Smartphone-Mimic Low-Luminance Blue Light Induces Retina Damage Through Bcl-2/BAX-Dependent Apoptosis.
2017; 157 (1): 196-210
Blue light-induced phototoxicity plays an important role in retinal degeneration and might cause damage as a consequence of smartphone dependency. Here, we investigated the effects of periodic exposure to blue light-emitting diode in a cell model and a rat retinal damage model. Retinal pigment epithelium (RPE) cells were subjected to blue light in vitro and the effects of blue light on activation of key apoptotic pathways were examined by measuring the levels of Bcl-2, Bax, Fas ligand (FasL), Fas-associated protein with death domain (FADD), and caspase-3 protein. Blue light treatment of RPE cells increased Bax, cleaved caspase-3, FasL, and FADD expression, inhibited Bcl-2 and Bcl-xL accumulation, and inhibited Bcl-2/Bax association. A rat model of retinal damage was developed with or without continuous or periodic exposure to blue light for 28 days. In this rat model of retinal damage, periodic blue light exposure caused fundus damage, decreased total retinal thickness, caused atrophy of photoreceptors, and injured neuron transduction in the retina.
View details for DOI 10.1093/toxsci/kfx030
View details for PubMedID 28184904
Long-term Fluorometholone Topical Use Induces Ganglion Cell Damage in Rats Analyzed With Optical Coherence Tomography
2015; 147 (2): 317-325
To determine the toxic effects of long-term topical usage of fluorometholone (FLM) on ganglion cells using a direct in vivo retinopathological Brown Norway (BN) rat model. The BN rat retinal model was investigated with a minimum of 3 rats and a maximum of 4 rats per group. Rats received vehicle and 0.02% FLM suspension via topical administration 3 times a day for 28 days. The fundus images and retinal vessels were detected on days 1, 14, and 28 using Micron III retinal imaging microscope and fundus fluorescein angiography (FFA). For retinal structures, spectral-domain optical coherence tomography (SD-OCT) images were taken after FFA on days 1, 14, and 28 using an SD-OCT Imaging System. For retinal function, electrical signal transduction of photoreceptors and bipolar cells was determined by electroretinographic (ERG) recording on days 1 and 28 and IOP detection. At the end of the experiment on day 28, immunohistochemistry and TUNEL assay were performed to investigate apoptosis in ganglion cells. Total retina and nerve fiber layer (NFL) to the inner plexiform layer (IPL) were significantly thinner following 28 days of FLM treatment. Hematoxylin and eosin stain showed that there were NFL and ganglion cell layer deformations in the FLM group. With FLM treatment, TUNEL assay showed approximately a 4.68-fold increase in apoptotic cells. Moreover, FLM decreased ERG b-wave amplitude by about 56%. Using ophthalmofundoscopy devices, after 28 days of topical administration, FLM decreased NFL-IPL and total retina thickness. This suggests that long-term FLM induces adverse effects with respect to ganglion cell apoptosis.
View details for DOI 10.1093/toxsci/kfv132
View details for Web of Science ID 000365547300005
View details for PubMedID 26141393
2,4,5-TMBA, a natural inhibitor of cyclooxygenase-2, suppresses adipogenesis and promotes lipolysis in 3T3-L1 adipocytes.
Journal of agricultural and food chemistry
2012; 60 (29): 7262-9
Obesity is a global health problem. Because of the high costs and side effects of obesity-treatment drugs, the potential of natural products as alternatives for treating obesity is under exploration. 2,4,5-Trimethoxybenzaldehyde (2,4,5-TMBA) present in plant roots, seeds, and leaves was reported to be a significant inhibitor of cyclooxygenase-2 (COX-2) activity at the concentration of 100 μg/mL. Because COX-2 is associated with differentiation of preadipocytes, the murine 3T3-L1 cells were cultured with 100 μg/mL of 2,4,5-TMBA during differentiation and after the cells were fully differentiated to study the effect of 2,4,5-TMBA on adipogenesis and lipolysis. Oil Red O staining and triglyceride assay revealed that 2,4,5-TMBA inhibited the formation of lipid droplets during differentiation; moreover, 2,4,5-TMBA down-regulated the protein levels of adipogenic signaling molecules and transcription factors MAP kinase kinase (MEK), extracellular signal-regulated kinase (ERK), CCAAT/enhancer binding protein (C/EBP)α, β, and δ, peroxisome proliferator-activated receptor (PPAR)γ, adipocyte determination and differentiation-dependent factor 1 (ADD1), and the rate-limiting enzyme for lipid synthesis acetyl-CoA carboxylase (ACC). In fully differentiated adipocytes, treatment with 2,4,5-TMBA for 72 h significantly decreased lipid accumulation by increasing the hydrolysis of triglyceride through suppression of perilipin A (lipid droplet coating protein) and up-regulation of hormone-sensitive lipase (HSL). The results of this in vitro study will pioneer future in vivo studies on antiobesity effects of 2,4,5-TMBA and selective COX-2 inhibitors.
View details for DOI 10.1021/jf302285k
View details for PubMedID 22746947