ABSTRACT
OBJECTIVE: To describe a novel scoring system of feline pigmented iris lesions prior to utilization of diode laser ablation of progressive pigmented iris lesions and to retrospectively evaluate short- and long-term patient outcomes following transcorneal diode laser ablation. ANIMALS: 317 client-owned cats (356 eyes) were included. CLINICAL PRESENTATION: Records of cats undergoing diode laser ablation from January 2000 to December 2018 were retrospectively reviewed. A novel clinical grading system to describe severity of feline iris hyperpigmentation was developed. Recorded parameters included signalment, operated-upon eye, presurgical iris pigmentation score, intraocular pressure, visual status, postoperative complications, repeat laser surgery, patient status at last follow-up, time to death, and presumptive or known cause of death. RESULTS: Complications included corneal ulceration (25/356 [7%]), glaucoma (18/356 [5%]), uveitis (4/356 [1.1%]), and corneal edema (3/356 [0.8%]). Enucleation was performed in 12 eyes due to blindness and secondary glaucoma. Repeat laser due to continued progression of pigment was performed in 18.5% of eyes. Two study patients were euthanized due to presumptive metastatic disease. Of the 250 cats for whom confirmation was available via phone call or medical records, 240 (96%) were alive at 1 year. CLINICAL RELEVANCE: Diode laser ablation appears safe overall and may be effective in decreasing progression of feline iris pigmentation. Complication risks appear minimal.
Subject(s)
Cat Diseases , Glaucoma , Laser Therapy , Cats , Animals , Retrospective Studies , Iris/surgery , Laser Therapy/veterinary , Glaucoma/veterinary , Eye Color , Intraocular Pressure , Cat Diseases/surgeryABSTRACT
PURPOSE: To survey commonly used, sterile ophthalmic viscoelastic materials used during routine cataract surgery for the presence of bacterial DNA and/or viable bacteria and endotoxin quantification. METHODS: Samples from three different ophthalmic viscoelastic manufacturers and three different production lots per manufacturer were collected for 16 S ribosomal ribonucleic acid (rRNA) sequencing and conventional aerobic and capnophilic bacterial culture. Other samples of viscoelastic material from the same three manufacturers were collected for endotoxin quantification using a commercially available Limulus amebocyte lysate (LAL) assay. Statistical analysis was performed using Sigma Plot 14.0, and R v4.0.2.0. Differences (p ≤ .05) between sample collection sites in total DNA concentration, microbial richness, mean intra-group distances, and endotoxin quantification alongside reagent controls were evaluated. RESULTS: Culture yielded two isolates, identified as Staphylococcus epidermidis and Bacillus megaterium. 16 S rRNA sequencing revealed no differences between brands in richness or overall composition. The most common bacterial DNA detected across all brands was Staphylococcus sp., Cutibacterium sp., Flavobacterium sp., and Lactobacillus sp. A significant difference was found between the median endotoxin concentration between Anvision and Hyvisc® viscoelastic (Anvision: 0.171 EU/mL, Hyvisc®: 0.03 EU/mL; p < .001). CONCLUSIONS: No brand-specific differences in bacterial DNA were detected in the viscoelastic materials. Staphylococcus, Cutibacterium, Flavobacterium, and Lactobacillus were the dominant contributors to the bacterial DNA detected. Although Anvision viscoelastic samples contained significantly more endotoxin than Hyvisc® viscoelastic samples, endotoxin concentrations were below the FDA limit of 0.2 EU/mL for both manufacturers. These data further the understanding of inflammatory outcomes following cataract surgery.
Subject(s)
Cataract , Hyaluronic Acid , Animals , DNA, Bacterial/genetics , Endotoxins/analysis , Bacteria , Cataract/veterinaryABSTRACT
Inflammatory outcomes, including toxic anterior segment syndrome (TASS) and infectious endophthalmitis, are potentially painful, blinding complications following cataract surgery. In an in vitro pilot study, commercially available, sterile foldable intraocular lenses (IOLs) used during routine canine cataract surgery, and their packaging fluid were surveyed for the presence of bacterial DNA and/or viable (cultivable) bacteria. Swabs from IOLs and packaging fluid from three different veterinary manufacturers and three different production lots/manufacturer were collected for 16S ribosomal ribonucleic acid (rRNA) sequencing. Packaging fluid samples were collected for aerobic/capnophilic bacterial culture. Culture yielded one isolate, identified as Staphylococcus epidermidis. 16S rRNA sequencing revealed distinct brand-specific bacterial DNA profiles, conserved between IOLs and packaging fluid of all production lots within each manufacturer. The dominant taxonomy differentiating each manufacturer was annotated as Staphylococcus sp, and was a 100% match to S. epidermidis. Distinct mixtures of bacterial DNA are present and consistent in IOLs and packaging fluid depending on the manufacturer, and Staphylococcus is the dominant contributor to the bacterial DNA detected. Caralens products had a significantly lower amount of Staphylococcus spp. compared to Anvision and Dioptrix products.
Subject(s)
Cataract , Lenses, Intraocular , Dogs , Animals , RNA, Ribosomal, 16S/genetics , DNA, Bacterial/genetics , Pilot Projects , Bacterial Adhesion , Staphylococcus epidermidis/genetics , Staphylococcus/geneticsABSTRACT
An array of corneal pathologies collectively called mustard gas keratopathy (MGK) resulting from ocular exposure to sulfur mustard (SM) gas are the most prevalent chemical warfare injury. MGK involves chronic ocular discomfort that results in vision impairment. The etiology of MGK remains unclear and poorly understood primarily due to a lack of scientific data regarding structural and cellular changes in different layers of the cornea altered by mustard vapor exposure in vivo. The goals of this study were to (a) characterize time-dependent changes in different layers of corneal epithelium, stroma, and endothelium in live animals in situ by employing state-of-the-art multimodal clinical ophthalmic imaging techniques and (b) determine if SM-induced acute changes in corneal cells could be rescued by a topical eye drop (TED) treatment using in an established rabbit in vivo model. Forty-five New Zealand White Rabbit eyes were divided into four groups (Naïve, TED, SM, and SM + TED). Only one eye was exposed to SM (200 mg-min/m3 for 8 min), and each group had three time points with six eyes each (Table-1). TED was topically applied twice a day for seven days. Clinical eye examinations and imaging were performed in live rabbits with stereo, Slit-lamp, HRT-RCM3, and Spectralis microscopy system. Fantes grading, fluorescein staining, Schirmer's tests, and applanation tonometry were conducted to measure corneal haze, ocular surface aberrations, tears, and intraocular pressure respectively. H&E and PSR staining were used for histopathological cellular changes in the cornea. In vivo confocal and OCT imaging revealed significant changes in structural and morphological appearance of corneal epithelium, stroma, and endothelium in vivo in SM-exposed rabbit corneas in a time-dependent manner compared to naïve cornea. Also, SM-exposed eyes showed loss of corneal transparency characterized by increased stromal thickness and light-scattering myofibroblasts or activated keratocytes, representing haze formation in the cornea. Neither naive nor TED-alone treated eyes showed any structural, cellular, and functional abnormalities. Topical TED treatment significantly reduced SM-induced abnormalities in primary corneal layers. We conclude that structural and cellular changes in primary corneal layers are early pathological events contributing to MGK in vivo, and efficient targeting of them with suitable agents has the potential to mitigate SM ocular injury.
Subject(s)
Burns, Chemical , Chemical Warfare Agents , Corneal Diseases , Mustard Gas , Rabbits , Animals , Mustard Gas/toxicity , Chemical Warfare Agents/toxicity , Cornea/pathology , Corneal Diseases/pathology , Burns, Chemical/pathology , Ophthalmic Solutions/pharmacology , FluoresceinsABSTRACT
Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts in vitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit in vivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24 h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n = 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p < 0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p < 0.001), fibronectin (3.2-fold; p < 0.001), collagen I (0.8-fold; p < 0.001), and collagen III (1.4-fold; p < 0.001), as well as protein levels of α-SMA (23.8%; p < 0.001) and collagens (1.8-fold; p < 0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-ß signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.
Subject(s)
Corneal Diseases , Corneal Injuries , Corneal Opacity , Actins/genetics , Alkalies , Animals , Cicatrix/pathology , Cicatrix/therapy , Cornea , Corneal Diseases/genetics , Corneal Diseases/therapy , Corneal Injuries/pathology , Corneal Injuries/therapy , Corneal Opacity/pathology , Corneal Opacity/therapy , Dependovirus , Fibronectins/genetics , Fibrosis , Genetic Therapy/methods , RNA, Messenger , Rabbits , Transforming Growth Factors/geneticsABSTRACT
Corneal injury and aberrant wound healing commonly result in corneal fibrosis and subsequent vision loss. Intermediate-conductance calmodulin/calcium-activated K+ channels (KCa3.1) have been shown to promote fibrosis in non-ocular and ocular tissues via upregulation of transforming growth factor beta (TGFß). TRAM-34 is a selective inhibitor of KCa3.1 and reduces fibrosis by downregulation of TGFß-induced transdifferentiation of stromal fibroblasts to myofibroblasts. Ascorbic acid has been demonstrated to be effective in promoting corneal re-epithelialization and reduction of neovascularization via anti-VEGF and anti-MMP mechanisms. This study evaluates tolerability and efficacy of a novel combination of TRAM-34 (25µM) and ascorbic acid (10%) topical treatment for corneal fibrosis using an established in vivo rabbit model and conducting clinical eye examinations. Markers of corneal fibrosis were evaluated in all corneas at study endpoint via histopathology, immunofluorescence, and quantitative real-time PCR. The eyedrop treated eyes showed significantly improved clinical outcomes based on modified McDonald Shadduck scores, reduction of clinical haze on Fantes scores, and reduction of central corneal thickness (CCT). At cellular and molecular levels, eyedrop treatment also significantly reduced expression of alpha smooth muscle actin (α-SMA) mRNA and protein, collagen III mRNA, and fibronectin mRNA compared to non-treated eyes. Our study suggests that a tested new bimodal eyedrop is well tolerated and effectively reduces corneal fibrosis/haze in rabbits in vivo.
Subject(s)
PyrazolesABSTRACT
A 10-year-old Boer goat wether presented for unilateral exophthalmos of 2- to 3-week duration. Ocular ultrasonography and computed tomography (CT) were utilized in the diagnosis of the patient's orbital disease and surgical planning. Exenteration was performed under the same general anesthetic event as CT. Cytology, culture, and histopathology were performed after exenteration. Cytology was consistent with a mixed bacterial infection. Culture confirmed the presence of Streptococcus ovis. Histopathology on the enucleated globe and mass revealed no evidence of tumor and confirmed intraocular extension of retrobulbar inflammation. Histopathologic diagnosis was consistent with severe chronic orbital pyogranuloma and fibrinosuppurative endophthalmitis confined to the subretinal space. The abscess recurred in the orbital space 2 weeks postoperatively; the orbit was explored. Repeat culture was consistent with S. ovis, Staphylococcus schleigeri subspecies coagulans, and Fusobacterium necrophorum. Complete resolution was obtained after drainage and lavage of the orbit. Abscess is cited as a cause of exophthalmos in small ruminants, but no individual case reports exist. Advanced imaging allowed presumptive diagnosis and surgical planning. Histopathology confirmed intraocular extension of retrobulbar disease.
Subject(s)
Exophthalmos , Goat Diseases , Orbital Diseases , Abscess/diagnosis , Abscess/surgery , Abscess/veterinary , Animals , Exophthalmos/diagnosis , Exophthalmos/etiology , Exophthalmos/veterinary , Goat Diseases/diagnosis , Goat Diseases/surgery , Goats , Male , Orbit , Orbital Diseases/diagnosis , Orbital Diseases/surgery , Orbital Diseases/veterinaryABSTRACT
Sulfur mustard (SM) is a chemical warfare agent that has been used throughout recent history and remains a threat today. Exposed soldiers and civilians experience a variety of symptoms primarily in the respiratory system, skin, and eyes. The ocular tissues are highly sensitive to damage by SM and undergo unique manifestations of acute, chronic, and delayed complications that can persist for months and years after exposure. The mechanisms of this unique mustard gas keratopathy are still not fully understood and animal models for the study of this disease are discussed. Recent advances in mechanisms of injury are included in this review. Ophthalmic manifestations of SM injury including persistent epithelial defects, limbal stem cell deficiency, corneal neovascularization, dry eye, and corneal opacification have been reported. A wide variety of medical and surgical therapies have been studied and are reviewed here along with potential future therapies.
Subject(s)
Chemical Warfare Agents/toxicity , Eye Diseases/chemically induced , Mustard Gas/toxicity , Drug Administration Schedule , HumansABSTRACT
PURPOSE: To describe the occurrence and associated factors for "fibrin web" (FW) formation following phacoemulsification in dogs. METHODS: A retrospective review of medical records of all dogs undergoing phacoemulsification (MU-Veterinary Health Center, 2014-2018) was conducted to associate FW formation with signalment, systemic co-morbidities, cataract stage, surgeon (resident vs faculty), phacoemulsification time, IOL, and intracameral injections including viscoelastic type. Both univariate and multivariate statistical analyses were performed to evaluate associations among variables with FW formation. RESULTS: Data from 398 eyes on 201 dogs were included; 4 left eyes (4 dogs) developed presumptive endophthalmitis and were excluded from further analysis. Forty-eight eyes did not have cataract surgery. Hence, 350 eyes on 201 dogs were included in the analyses. Among these, 84 eyes (59 dogs) developed a FW. Univariate analyses showed that the odds of FW increased with age and phacoemulsification time. Additionally, FW web was associated lens type, lens brand, and viscoelastic type. Multivariate analyses showed that when comparing lens types in combination with a particular viscoelastic, viscoelastic impacted the estimated prevalence of FW formation the most. In contrast, when the data were analyzed by lens brand, lens brand impacted prevalence more than viscoelastic type. Diabetes mellitus was not associated with FW formation. CONCLUSIONS: Based on the available data, intraocular lens implantation, viscoelastic type, dog age, and phacoemulsification time were associated with FW formation. Diabetes mellitus, gender, cataract stage, surgeon, intracameral injections other than viscoeleastic, and intra- and postoperative complications were not associated with FW formation.
Subject(s)
Cataract/veterinary , Fibrin/metabolism , Phacoemulsification/veterinary , Uveitis, Anterior/veterinary , Animals , Cataract/metabolism , Dogs , Female , Male , Phacoemulsification/adverse effects , Retrospective Studies , Uveitis, Anterior/etiologyABSTRACT
Next-generation sequencing (NGS) methods have been used to identify a diverse ocular surface (OS) microbiota in humans. These results have highlighted limitations in microbial detection via traditional culture-based techniques. The OS has mechanisms such as tear film and mechanical blinking, which may aid in preventing adherence and colonization of microbes, suggesting that only low populations of microbes may reside on the OS. Additionally, closely related tissues to the OS are exposed to a similar array of microbes, but demonstrate different defense mechanisms. Information regarding concordance of microbial communities of the OS and nearby tissues is lacking. Our study purposes were to (1) characterize the conjunctival microbiota of healthy dogs, (2) compare the conjunctival microbiota to the periocular haired skin and distal nose, and (3) compare the bacteria identified by culture to NGS of the healthy canine conjunctiva. Here, NGS was used to evaluate samples from 25 healthy adult dogs of the conjunctiva, periocular haired skin, and distal nose. Additional samples were collected from each dog for traditional conjunctival culture. The 16S rRNA gene amplicon libraries were evaluated for coverage, relative abundance, richness, and diversity. Site-dependent similarities evaluated using principal coordinate analysis (PCoA) and PERMANOVA demonstrated relatedness in community compositions between sites. The conjunctiva of healthy dogs yielded a rich and diverse microbiota based on NGS. While some regional continuity was noted, microbial communities of the conjunctiva, periocular haired skin, and nose were significantly different from each other. Comparatively, traditional culture markedly underestimated the number of bacterial taxa present on the healthy canine OS. Findings suggest similarities in nasal and conjunctival microbial communities, which may be a result of similarities in mucosal immunity and anatomic connection via the nasolacrimal system. Further investigation using NGS into changes of the composition of bacterial communities in disease is warranted.
ABSTRACT
Wound healing differs significantly between men and women in a tissue-dependent manner. Dermal wounds heal faster in women whereas mucosal wounds heal faster in men. However, the effect of sex as a variable in corneal wound healing is largely unknown. The primary objective of this study was to test whether sex is a biological variable in corneal wound healing activated by the trauma or injury using an established in vivo rabbit model with male and female New Zealand White rabbits. Corneal wounds in rabbits were produced by a single topical alkali (0.5N Sodium hydroxide) application. Serial slit-lamp, stereo biomicroscopy, and applanation tonometry evaluated corneal opacity, anterior segment ocular health, and intraocular pressure (IOP), respectively, at various times during the study. Fourteen days after alkali-wound, corneal tissues were collected after humane euthanasia to examine cellular and molecular wound healing parameters. Quantitative PCR (qPCR) and immunofluorescence were used to quantify changes in the extracellular modeling protein levels of alpha-smooth muscle actin (α-SMA), Fibronectin (FN), Collagen-I (Col-I), and Transforming growth factor beta 1 (TGFß1) involved in corneal healing. Hematoxylin and Eosin (H&E) staining was used to study histopathological changes in morphology and TUNEL assay to evaluate levels of apoptotic cell death. Male and female rabbits showed no significant differences in corneal opacity (Fantes score) or intraocular pressure (IOP) values (9.5⯱â¯0.5â¯mm Hg) in live animals. Likewise, no statistically significant sex-based differences in the mRNA levels of α-SMA (maleâ¯=â¯5.95⯱â¯0.21 fold vs. femaleâ¯=â¯5.32⯱â¯0.043), FN (maleâ¯=â¯3.02⯱â¯0.24 fold vs. femaleâ¯=â¯3.23⯱â¯0.27), Col-I (maleâ¯=â¯3.12⯱â¯0.37 fold vs. femaleâ¯=â¯3.31⯱â¯0.24), TGFß1 (maleâ¯=â¯1.65⯱â¯0.06 fold vs. femaleâ¯=â¯1.59⯱â¯0.053); and protein levels of α-SMA (maleâ¯=â¯74.16⯱â¯4.6 vs. femaleâ¯=â¯71.58⯱â¯7.1), FN (maleâ¯=â¯60.11⯱â¯4.6 vs. femaleâ¯=â¯57.41⯱â¯8.3), Col-I (maleâ¯=â¯84.11⯱â¯2.8 vs. femaleâ¯=â¯84.55⯱â¯3.6), TGFß1 (maleâ¯=â¯11.61⯱â¯2.8 vs. femaleâ¯=â¯9.5⯱â¯3.04) were observed. Furthermore, H&E and TUNEL analyses found no statistically significant differences in cellular structures and apoptosis, respectively, in male vs. female corneas. Consistent with earlier reports, wounded corneas showed significantly increased levels of these parameters compared to the unwounded corneas. Our data suggest that sex is not a major biological variable during active early stages of corneal wound healing in rabbits in vivo.
Subject(s)
Burns, Chemical/physiopathology , Corneal Injuries/physiopathology , Eye Burns/chemically induced , Sex Factors , Wound Healing/physiology , Actins/genetics , Animals , Burns, Chemical/genetics , Collagen Type I/genetics , Corneal Injuries/genetics , Eye Burns/genetics , Eye Burns/physiopathology , Fibronectins/genetics , Fluorescent Antibody Technique , In Situ Nick-End Labeling , RNA, Messenger/genetics , Rabbits , Real-Time Polymerase Chain Reaction , Sodium Hydroxide/toxicity , Transforming Growth Factor beta1/geneticsABSTRACT
Ocular pathogens cause many painful and vision-threatening diseases such as infectious keratitis, uveitis, and endophthalmitis. While virulent pathogens and pathobionts play important roles in disease pathogenesis, the scientific community has long assumed disruption of the ocular surface occurs prior to microbial colonization and subsequent infection. While nonpathogenic bacteria are often detected in corneal and conjunctival cultures from healthy eyes, cultures also frequently fail to yield growth of common ocular pathogens or nonpathogenic bacteria. This prompts the following question: Is the ocular surface populated by a stable microbial population that cannot be detected using standard culture techniques? The study of the microbiome has recently become a widespread focus in physician and veterinary medicine. Research suggests a pivotal symbiotic relationship with these microbes to maintain healthy host tissues, and when altered is associated with various disease states ("dysbiosis"). The microbiota that lives within and on mammalian bodies have long been known to influence health and susceptibility to infection. However, limitations of traditional culture methods have resulted in an incomplete understanding of what many now call the "forgotten organ," that is, the microbiome. With the introduction of high-throughput sequencing, physician ophthalmology has recognized an ocular surface with much more diverse microbial communities than suspected based on traditional culture. This article reviews the salient features of the ocular surface microbiome and highlights important future applications following the advent of molecular techniques for microbial identification, including characterizing ocular surface microbiomes in our veterinary species and their potential role in management of infectious and inflammatory ocular diseases.
Subject(s)
Eye/microbiology , Microbiota , Animals , Bacteria/classification , Bacteria/isolation & purification , Humans , Molecular TypingABSTRACT
OBJECTIVES: To serially evaluate morphologic and elemental composition changes to diamond burr tips (DBTs) comparing two sterilization protocols. ANIMALS STUDIED: A total of 300 fresh cadaver porcine globes. PROCEDURES: Six DBTs were randomly, equally assigned into Group 1 or 2, and then analyzed using Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) at 0, 25, 50, and 100 cycles. Diamond burr debridement (DBD) was performed for 120 seconds on corneal stroma using the Algerbrush®. DBTs were cleaned, and then: Group 1 was sterilized by Germinator 500™; and Group 2 underwent ultrasonic cleaning and pre-vacuum autoclave. A cycle is defined as one DBD, cleaning and sterilization protocol. Data were quantified using custom MatLab program. RESULTS: Energy Dispersive Spectroscopy revealed minor buildup of sulfur on both groups. Group 1 displayed major buildup of carbon and calcium. All DBTs were stippled with inorganic particulate at baseline. Particulates were no longer present on Group 2 by 25 cycles, but remained on Group 1 at all time points. There was significantly more buildup on Group 1 at all time points (P = 0.0000, 0.0009, and 0.0003 for 25, 50, and 100 cycles, respectively). More damage to Group 2 at all time points (P = 0.003, 0.002, and 0.003 for 25, 50, and 100 cycles, respectively) was observed. CONCLUSIONS: No significant damage to Group 1 DBTs was noted after 100 cycles, however, particulate matter is not adequately removed using this sterilization technique. Ultrasonic cleaning is warranted between DBDs to achieve adequate particulate removal prior to sterilization; greater damage occurs with this technique which supports replacing DBTs regularly.
Subject(s)
Debridement/veterinary , Sterilization/methods , Animals , Debridement/instrumentation , Diamond , Dogs , Equipment Contamination , Microscopy, Electron, Scanning , Random Allocation , Spectrum Analysis , UltrasonicsABSTRACT
PURPOSE: This study investigated the efficiency and potential toxicity of a linear 22-kDa polyethylenimine (PEI)-DNA nanoconstruct for delivering genes to corneal cells and the effects of PEI nitrogen-to-DNA phosphate (N:P) ratio on gene transfer efficiency in vitro and in vivo. METHODS: A gel retardation assay, zeta potential measurement, bright-field microscopy, transfection with green fluorescent protein (GFP), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were used to characterize the physicochemical and biological properties and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and reactive oxygen species (ROS) assay for cytotoxicity of the linear PEI-DNA nanoconstruct using in vitro cultured primary human corneal fibroblast and in vivo mouse models. RESULTS: Of the several evaluated N:P ratios, the highest gene transfection efficiency achieved without any notable cytotoxicity was observed at an N:P ratio of 30:1 (N:P 30). In vivo gene transfer studies revealed substantial GFP gene delivery into the corneas of mice 3 days after a single 5-min topical application without any significant adverse ocular effects. Slit-lamp biomicroscope ophthalmic examination of the mouse exposed to the linear PEI-DNA nanoconstruct showed no evidence of hyperemia (redness), corneal edema, ocular inflammation, or epiphora (excessive tearing). CONCLUSIONS: The 22-kDa linear PEI-DNA nanoconstruct is an efficient and well-tolerated vector for corneal gene therapy in vitro and in vivo and could be used as a platform for developing novel gene-based nanomedicine approaches for corneal diseases.
Subject(s)
Cornea/metabolism , DNA/chemistry , Gene Transfer Techniques , Nanoparticles/chemistry , Polyethyleneimine/chemistry , Animals , Cell Survival/drug effects , Cells, Cultured , Cornea/drug effects , DNA/administration & dosage , DNA/pharmacology , Female , Genetic Vectors/administration & dosage , Genetic Vectors/chemistry , Genetic Vectors/pharmacology , Humans , Mice , Mice, Inbred C57BL , Nanoparticles/administration & dosage , Optical Imaging , Particle Size , Polyethyleneimine/administration & dosage , Polyethyleneimine/pharmacologyABSTRACT
Decorin (Dcn), a small leucine-rich proteoglycan, is involved in the regulation of corneal wound healing. Epidermal growth factor receptor (EGFR) plays a critical role in corneal fibroblasts proliferation, migration and extracellular matrix (ECM) modulation upon injury or infection. The present study aimed to investigate the mechanistic role of Dcn in EGFR internalization to the regulation of corneal stromal fibroblasts (CSFs) migration, a key step in the corneal wound healing. Human corneal stromal fibroblasts (hCSF) cultures were generated from donor corneas. At 70% confluence, cells were switched to serum-free conditions for 48â¯h and then treated with decorin (250â¯nM) in the presence or absence of EGF (100â¯ng/ml) for various time points (10-60â¯min). Cell lysates were subjected to proteome array analysis screening for 42 different phosphorylated human receptor tyrosine kinases (RTKs), immunocytochemistry, and western blots to analyze EGFR phosphorylation. The scratch-wound assay was performed to evaluate the effects of decorin on EGF-mediated hCSF migration. Dcn caused a rapid EGFR phosphorylation within 10â¯min of exposure in RTK blot defining its role as a biological ligand for EGFR in hCSFs. Prolonged exposure to Dcn caused complete disappearance of EGFR and inhibition of the hCSF migration in the scratch wound assay suggesting Dcn binding to EGFR causes EGFR down-regulation. Immunostaining studies indicated that Dcn-treatment to hCSFs internalizes Dcn-EGFR complex, which does not require tyrosine kinase activity when treated with the AG1478 inhibitor and co-localizes the complex to the perinuclear region. Next, we found that Dcn-EGFR complex does not follow canonical early endosome internalization as revealed by the EEA1 antibody instead binds to the CD63 antibody directed for degradation by the late endosome. We also found that Dcn regulates the EGFR recycling by preventing its binding to Rab11, a specific antibody for recycling endosome. Further, hCSFs-pretreated with pharmacological inhibitors, methyl-ß-cyclodextrin and chlorpromazine and supplemented with Dcn suggested EGFR trafficking via the caveolae-mediated pathway. These results suggest that Dcn acts as a biological ligand for EGFR and modulates hCSF migration via EGFR down-regulation, thus playing a vital role in corneal wound healing.
Subject(s)
Caveolae/metabolism , Cell Movement/physiology , Corneal Keratocytes/physiology , Decorin/physiology , Endocytosis/physiology , ErbB Receptors/metabolism , Adult , Aged , Blotting, Western , Cells, Cultured , Corneal Stroma/cytology , Decorin/pharmacology , Epidermal Growth Factor/pharmacology , Female , Humans , Immunohistochemistry , Male , Middle Aged , Phosphorylation , Proteomics , Receptor Protein-Tyrosine Kinases/metabolism , Young AdultABSTRACT
Purpose: We tested the potential of bone morphogenic protein 7 (BMP7) and hepatocyte growth factor (HGF) combination gene therapy to treat preformed corneal fibrosis using established rabbit in vivo and human in vitro models. Methods: Eighteen New Zealand White rabbits were used. Corneal fibrosis was produced by alkali injury. Twenty-four hours after scar formation, cornea received topically either balanced salt solution (BSS; n = 6), polyethylenimine-conjugated gold nanoparticle (PEI2-GNP)-naked plasmid (n = 6) or PEI2-GNP plasmids expressing BMP7 and HGF genes (n = 6). Donor human corneas were used to obtain primary human corneal fibroblasts and myofibroblasts for mechanistic studies. Gene therapy effects on corneal fibrosis and ocular safety were evaluated by slit-lamp microscope, stereo microscopes, quantitative real-time PCR, immunofluorescence, TUNEL, modified MacDonald-Shadduck scoring system, and Draize tests. Results: PEI2-GNP-mediated BMP7+HGF gene therapy significantly decreased corneal fibrosis in live rabbits in vivo (Fantes scale was 0.6 in BMP7+HGF-treated eyes compared to 3.3 in -therapy group; P < 0.001). Corneas that received BMP7+HGF demonstrated significantly reduced mRNA levels of profibrotic genes: α-SMA (3.2-fold; P < 0.01), fibronectin (2.3-fold, P < 0.01), collagen I (2.1-fold, P < 0.01), collagen III (1.6-fold, P < 0.01), and collagen IV (1.9-fold, P < 0.01) compared to the -therapy corneas. Furthermore, BMP7+HGF-treated corneas showed significantly fewer myofibroblasts compared to the -therapy controls (83%; P < 0.001). The PEI2-GNP introduced >104 gene copies per microgram DNA of BMP7 and HGF genes. The recombinant HGF rendered apoptosis in corneal myofibroblasts but not in fibroblasts. Localized topical BMP7+HGF therapy showed no ocular toxicity. Conclusions: Localized topical BMP7+HGF gene therapy treats corneal fibrosis and restores transparency in vivo mitigating excessive healing and rendering selective apoptosis in myofibroblasts.
Subject(s)
Apoptosis/drug effects , Bone Morphogenetic Protein 7/genetics , Corneal Opacity/therapy , Genetic Therapy/methods , Hepatocyte Growth Factor/genetics , Myofibroblasts/pathology , Administration, Ophthalmic , Animals , Cornea/pathology , Corneal Opacity/pathology , Disease Models, Animal , Drug Combinations , Female , Fibrosis/therapy , Gold/chemistry , In Situ Nick-End Labeling , Intraocular Pressure , Metal Nanoparticles/chemistry , Plasmids/genetics , Polyethyleneimine/chemistry , Rabbits , Real-Time Polymerase Chain Reaction , Tonometry, OcularABSTRACT
OBJECTIVE: To explore the impact of equine corneal fibroblast (ECF) to myofibroblast (ECM) differentiation by altering the expression of the Smad genes either individually or in combination. Specifically, we sought to examine the ECF differentiation after (a) silencing of Smad2, 3, and 4 profibrotic genes individually and (b) overexpression of antifibrotic Smad7 gene and in a combination with pro- and antifibrotic Smad genes. METHODS: Equine corneal fibroblast primary cultures were generated as previously described. ECFs were transfected with individual plasmids which silenced gene expression of either Smad2, 3, or 4 or in combination with a plasmid overexpressing Smad7 using Lipofectamine 2000™ or Lipofectamine BLOCK-iT™. Smad-transfected clones were then exposed to TGF-ß1 to induce differentiation to myofibroblasts. Immunofluorescence and qRT-PCR techniques quantified levels of ECF differentiation to ECM by measuring alpha smooth muscle actin, a known marker of ECM transdifferentiation. RESULTS: Silencing of individual Smad2, 3, or 4 genes or overexpression of Smad7 showed significant inhibition of ECF transdifferentiation (73-83% reduction). Silencing of Smad2 showed the greatest inhibition of ECF transdifferentiation in (a) and was therefore utilized for the combination gene transfer testing. The combination gene transfer consisting of Smad7 overexpression and Smad2 silencing attenuated ECF differentiation significantly; however, the level was not significant compared to the overexpression of Smad7 individually. CONCLUSIONS: Using gene transfer technology involving profibrotic Smad silencing, antifibrotic Smad overexpression or its combination is a novel strategy to control TGF-ß1-mediated fibrosis in equine fibroblasts. Combination gene therapy was not better than single gene therapy in this study.
Subject(s)
Cell Differentiation/genetics , Cornea/cytology , Fibroblasts/cytology , Horses , Myofibroblasts/cytology , Smad Proteins/genetics , Animals , Cells, Cultured , Fibrosis/genetics , Fibrosis/therapy , Fibrosis/veterinary , Gene Silencing , Gene Transfer Techniques , Genetic Therapy/veterinary , RNA, Messenger/antagonists & inhibitors , Smad Proteins/economicsABSTRACT
An eleven-year-old, female spayed Boxer dog was diagnosed with a uveal schwannoma (formerly known as the spindle cell tumor of the blue-eyed dog or SCTBED) despite having a uniformly brown iris. The patient presented to emergency for ocular discomfort, and the right globe was subsequently enucleated due to glaucoma and submitted for histopathology. Upon histopathologic evaluation, a uveal schwannoma was diagnosed and confirmed with immunohistochemical staining. Complete metastatic evaluation 1 and 6 months after initial presentation did not reveal evidence of metastasis, and the dog remains systemically healthy. This case represents a unique variant of uveal schwannoma and is relevant because although the vast majority of these tumors occur in blue-eyed dogs, clinicians should not completely rule out this tumor as a differential based on the iris color.
Subject(s)
Neurilemmoma/veterinary , Uveal Neoplasms/pathology , Uveal Neoplasms/veterinary , Animals , Dog Diseases/pathology , Dogs , Eye Color , Female , Neurilemmoma/pathologyABSTRACT
OBJECTIVE To evaluate the coding regions of ADAMTS17 for potential mutations in Chinese Shar-Pei with a diagnosis of primary open-angle glaucoma (POAG), primary lens luxation (PLL), or both. ANIMALS 63 Shar-Pei and 96 dogs of other breeds. PROCEDURES ADAMTS17 exon resequencing was performed on buccal mucosal DNA from 10 Shar-Pei with a diagnosis of POAG, PLL, or both (affected dogs). A candidate causal variant sequence was identified, and additional dogs (53 Shar-Pei [11 affected and 42 unaffected] and 95 dogs of other breeds) were genotyped for the variant sequence by amplified fragment length polymorphism analysis. Total RNA was extracted from ocular tissues of 1 affected Shar-Pei and 1 ophthalmologically normal Golden Retriever; ADAMTS17 cDNA was reverse transcribed and sequenced, and ADAMTS17 expression was evaluated by quantitative reverse-transcription PCR assay. RESULTS All affected Shar-Pei were homozygous for a 6-bp deletion in exon 22 of ADAMTS17 predicted to affect the resultant protein. All unaffected Shar-Pei were heterozygous or homozygous for the wild-type allele. The variant sequence was significantly associated with affected status (diagnosis of POAG, PLL, or both). All dogs of other breeds were homozygous for the wild-type allele. The cDNA sequencing confirmed presence of the expected variant mRNA sequence in ocular tissue from the affected dog only. Gene expression analysis revealed a 4.24-fold decrease in the expression of ADAMTS17 in ocular tissue from the affected dog. CONCLUSIONS AND CLINICAL RELEVANCE Results supported that the phenotype (diagnosis of POAG, PLL, or both) is an autosomal recessive trait in Shar-Pei significantly associated with the identified mutation in ADAMTS17.