Medicine and Health Sciences Commons^™

Vorinostat: A Potent Agent To Prevent And Treat Laser-Induced Corneal Haze, Ashish Tandon, Jonathan C. K. Tovey, Michael R. Waggoner, Ajay Sharma, John W. Cowden, Daniel J. Gibson, Yuanjing Liu, Gregory S. Schultz, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

PURPOSE—This study investigated the efficacy and safety of vorinostat, a deacetylase (HDAC) inhibitor, in the treatment of laser-induced corneal haze following photorefractive keratectomy (PRK) in rabbits in vivo and transforming growth factor beta 1 (TGFβ1) -induced corneal fibrosis in vitro.

METHODS—Corneal haze in rabbits was produced with −9.00 diopters (D) PRK. Fibrosis in cultured human and rabbit corneal fibroblasts was activated with TGFβ1. Vorinostat (25 μm) was topically applied once for 5 minutes on rabbit cornea immediately after PRK for in vivo studies. Vorinostat (0 to 25 μm) was given to human/rabbit corneal fibroblasts for 5 minutes or 48 …

Attenuation Of Corneal Myofibroblast Development Through Nanoparticle-Mediated Soluble Transforming Growth Factor-Β Type Ii Receptor (Stgfβrii) Gene Transfer, Ajay Sharma, Jason T. Rodier, Ashish Tandon, Alexander M. Klibanov, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

Purpose: To explore (i) the potential of polyethylenimine (PEI)-DNA nanoparticles as a vector for delivering genes into human corneal fibroblasts, and (ii) whether the nanoparticle-mediated soluble extracellular domain of the transforming growth factor–β type II receptor (sTGFβRII) gene therapy could be used to reduce myofibroblasts and fibrosis in the cornea using an in vitro model.

Methods: PEI-DNA nanoparticles were prepared at a nitrogen-to-phosphate ratio of 30 by mixing linear PEI and a plasmid encoding sTGFβRII conjugated to the fragment crystallizable (Fc) portion of human immunoglobulin. The PEI-DNA polyplex formation was confirmed through gel retardation assay. Human corneal fibroblasts (HCFs) were …

Neuroprotective Effects Of Bilobalide Are Accompanied By A Reduction Of Ischemia-Induced Glutamate Release In Vivo, Dorothee Lang, Cornelia Kiewert, Alexander Mdzinarishvili, Tina Maria Schwarzkopf, Rachita K. Sumbria, Joachim Hartmann, Jochen Klein

Pharmacy Faculty Articles and Research

Neuroprotective properties of bilobalide, a specific constituent of Ginkgo extracts, were tested in a mouse model of stroke. After 24 h of middle cerebral artery occlusion (MCAO), bilobalide reduced infarct areas in the core region (striatum) by 40–50% when given at 10 mg/kg 1 h prior to MCAO. Neuroprotection was also observed at lower doses, or when the drug was given 1 h past stroke induction. Sensorimotor function in mice was improved by bilobalide as shown by corner and chimney tests. When brain metabolism in situ was monitored by microdialysis, MCAO caused a rapid disappearance of extracellular glucose in the …

Unifying The Mathematical Modeling Of In Vivo And In Vitro Microdialysis, Peter M. Bungay, Rachita K. Sumbria, Ulrich Bickel

Pharmacy Faculty Articles and Research

A unifying approach is presented for developing mathematical models of microdialysis that are applicable to both in vitro and in vivo situations. Previous models for cylindrical probes have been limited by accommodating analyte diffusion through the surrounding medium in the radial direction only, i.e., perpendicular to the probe axis, or by incomplete incorporation of diffusion in the axial direction. Both radial and axial diffusion are included in the present work by employing two-dimensional finite element analysis. As in previous models, the nondimensional clearance modulus (Θ) represents the degree to which analyte clearance from the external medium influences diffusion through the …

Polyethylenimine-Conjugated Gold Nanoparticles: Gene Transfer Potential And Low Toxicity In The Cornea, Ajay Sharma, Ashish Tandon, Jonathan C. K. Tovey, Rangan Gupta, J. David Robertson, Jennifer A. Fortune, Alexander M. Klibanov, John W. Cowden, Frank G. Rieger, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

This study examined the gene transfer efficiency and toxicity of 2-kDa polyethylenimine conjugated to gold nanoparticles (PEI2-GNP) in the human cornea in vitro and rabbit cornea in vivo. PEI2-GNP with nitrogen-to-phosphorus (N/P) ratios of up to 180 exhibited significant transgene delivery in the human cornea without altering the viability or phenotype of these cells. Similarly, PEI2-GNP applied to corneal tissues collected after 12 h, 72 h, or 7 days exhibited appreciable gold uptake throughout the rabbit stroma with gradual clearance of GNP over time. Transmission electron microscopy detected GNP in the keratocytes and the extracellular matrix of the rabbit corneas. …

Significant Inhibition Of Corneal Scarring In Vivo With Tissue-Selective, Targeted Aav5 Decorin Gene Therapy, Rajiv R. Mohan, Ashish Tandon, Ajay Sharma, John W. Cowden, Jonathan C. K. Tovey

Pharmacy Faculty Articles and Research

PURPOSE. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the stroma with adeno-associated virus serotype 5 (AAV5) inhibits corneal fibrosis in vivo without significant side effects.

METHODS. An in vivo rabbit model of corneal fibrosis was used. Targeted decorin gene therapy was delivered to the rabbit cornea by a single topical application of AAV5 (100 L; 6.5 1012 g/mL) onto the bare stroma for 2 minutes. The levels of corneal fibrosis were determined with stereomicroscopy, slit lamp biomicroscopy, -smooth muscle actin ( SMA), fibronectin, and F-actin immunocytochemistry, and/or immunoblotting. CD11b, F4/80 immunocytochemistry, and TUNEL assay …

Efficacious And Safe Tissue-Selective Controlled Gene Therapy Approaches For The Cornea, Rajiv R. Mohan, Sunilima Sinha, Ashish Tandon, Rangan Gupta, Jonathan C. K. Tovey, Ajay Sharma

Pharmacy Faculty Articles and Research

Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5), and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.561012 vg/ml) expressing green fluorescent protein gene (GFP) was topically applied onto normal or diseased (fibrotic or neovascularized) rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit …

Targeted Decorin Gene Therapy Delivered With Adeno-Associated Virus Effectively Retards Corneal Neovascularization In Vivo, Rajiv R. Mohan, Jonathan C. K. Tovey, Ajay Sharma, Gregory S. Schultz, John W. Cowden, Ashish Tandon

Pharmacy Faculty Articles and Research

Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 μl; 5x10^12 vg/ml) application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and …

Gene Delivery In The Equine Cornea: A Novel Therapeutic Strategy, Dylan G. Buss, Ajay Sharma, Elizabeth A. Giuliano, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

Objective—To determine if hybrid adeno-associated virus serotype 2/5 (AAV5) vector can effectively deliver foreign genes into the equine cornea without causing adverse side effects. The aims of this study were to: (i) evaluate efficacy of AAV5 to deliver therapeutic genes into equine corneal fibroblasts (ECFs) using enhanced green fluorescent protein (EGFP) marker gene and (ii) establish the safety of AAV5 vector for equine corneal gene therapy.

Animal Material—Primary ECF cultures were harvested from healthy donor equine corneas. Cultures were maintained at 370C in humidified atmosphere with 5% CO2.

Procedure—AAV5 vector expressing EGFP under control of hybrid cytomegalovirus (CMV) + chicken …

Vector Delivery Technique Affects Gene Transfer In The Cornea In Vivo, Rajiv R. Mohan, Ajay Sharma, Tyler C. Cebulko, Ashish Tandon

Pharmacy Faculty Articles and Research

Purpose: This study tested whether controlled drying of the cornea increases vector absorption in mouse and rabbit corneas in vivo and human cornea ex vivo, and studied the effects of corneal drying on gene transfer, structure and inflammatory reaction in the mouse cornea in vivo.

Methods: Female C57 black mice and New Zealand White rabbits were used for in vivo studies. Donor human corneas were used for ex vivo experiments. A hair dryer was used for drying the corneas after removing corneal epithelium by gentle scraping. The corneas received no, once, twice, thrice, or five times warm air for …

Isolation And Cultivation Of Equine Corneal Keratocytes, Fibroblasts And Myofibroblasts, Dylan G. Buss, Elizabeth A. Giuliano, Ajay Sharma, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

Objective—To establish an in vitro model for the investigation of equine corneal wound healing. To accomplish this goal, a protocol to isolate and culture equine corneal keratocytes, fibroblasts and myofibroblasts was developed.

Animal material—Equine corneal buttons were aseptically harvested from healthy research horses undergoing humane euthanasia for reasons unrelated to this study. Slit-lamp biomicroscopy was performed prior to euthanasia by a board-certified veterinary ophthalmologist to ensure that all samples were harvested from horses free of anterior segment disease.

Procedure—Equine corneal stroma was isolated using mechanical techniques and stromal subsections were then cultured. Customized media at different culture conditions was used …

Efficacy And Safety Of Mitomycin C As An Agent To Treat Corneal Scarring In Horses Using An In Vitro Model, Dylan G. Buss, Ajay Sharma, Elizabeth A. Giuliano, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

Objective—Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in-vitro model.

Procedure—An in-vitro model of equine corneal fibroblast (ECF) developed was used. The equine corneal fibroblast or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta-1 (TGFβ1) under serum-free conditions. The MMC dose for the equine cornea was defined with dose-dependent trypan blue exclusion and MTT [(3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays …

Localization Of Angiotensin Converting Enzyme In Rabbit Cornea And Its Role In Controlling Corneal Angiogenesis In Vivo, Ajay Sharma, Daniel I. Bettis, John W. Cowden, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

Purpose: The renin angiotensin system (RAS) has been shown to modulate vascular endothelial growth factor and angiogenesis. In this study we investigated (i) the existence of the RAS components angiotensin converting enzyme (ACE) and angiotensin II receptors (AT1 and AT2) in the rabbit cornea using in vitro and ex vivo models and (ii) the effect of enalapril, an ACE inhibitor, to inhibit angiogenesis in rabbit cornea in vivo.

Methods: New Zealand White rabbits were used. Cultured corneal fibroblasts and corneal epithelial cells were used for RNA isolation and cDNA preparation using standard molecular biology techniques. PCR was performed to …

Aav Serotype Influences Gene Transfer In Corneal Stroma In Vivo, Ajay Sharma, Jonathan C. K. Tovey, Arkasubhra Ghosh, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

This study evaluated the cellular tropism and relative transduction efficiency of three AAV serotypes, AAV6, AAV8 and AAV9, for corneal gene delivery using mouse cornea in vivo and donor human cornea ex vivo. The AAV6, AAV8 and AAV9 serotypes having AAV2 plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Viral vectors (109 vg/μl) were topically applied onto mouse cornea in vivo and human cornea ex vivo after removing the epithelium. Human corneas were processed for transgene delivery at day 5 after viral vector application. Mouse corneas were harvested …

Trichostatin A Inhibits Corneal Haze In Vitro And In Vivo, Ajay Sharma, Maneesh M. Mehan, Sunilima Sinha, John W. Cowden, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

PURPOSE. Trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to suppress TGF- –induced fibrogenesis in many nonocular tissues. The authors evaluated TSA cytotoxicity and its antifibrogenic activity on TGF- –driven fibrosis in the cornea with the use of in vitro and in vivo models.

METHODS. Human corneal fibroblasts (HSFs) were used for in vitro studies, and New Zealand White rabbits were used for in vivo studies. Haze in the rabbit cornea was produced with photorefractive keratectomy (PRK) using excimer laser. Trypan blue exclusion and MTT assays evaluated TSA cytotoxicity to the cornea. Density of haze in the rabbit …

Stromal Haze, Myofibroblasts, And Surface Irregularity After Prk, Marcelo V. Netto, Rajiv R. Mohan, Sunilima Sinha, Ajay Sharma, William Dupps, Steven E. Wilson

Pharmacy Faculty Articles and Research

The aim of this study was to investigate the relationship between the level of stromal surface irregularity after photorefractive keratectomy (PRK) and myofibroblast generation along with the development of corneal haze.

Variable levels of stromal surface irregularity were generated in rabbit corneas by positioning a fine mesh screen in the path of excimer laser during ablation for a variable percentage of the terminal pulses of the treatment for myopia that does not otherwise generate significant opacity. Ninety-six rabbits were divided into eight groups[.]

Slit lamp analysis and haze grading were performed in all groups. Rabbits were sacrificed at 4 hr …

Effect Of Prophylactic And Therapeutic Mitomycin C On Corneal Apoptosis, Cellular Proliferation, Haze, And Long-Term Keratocyte Density In Rabbits, Marcelo V. Netto, Rajiv R. Mohan, Sunilima Sinha, Ajay Sharma, Pankaj C. Gupta, Steven E. Wilson

Pharmacy Faculty Articles and Research

PURPOSE—To determine the mechanism through which topical mitomycin C prevents and treats corneal haze after photorefractive keratectomy (PRK) and to examine the effects of dosage and duration of exposure.

METHODS—In 224 New Zealand rabbits, −9.0 diopter PRK with mitomycin C or balanced salt solution was performed. Haze level was graded at the slit-lamp. Rabbits were sacrificed at 4 hours, 24 hours, 4 weeks, or 6 months after surgery and immunohistochemistry was performed with TUNEL assay, Ki67 and α-SMA.

RESULTS—TUNEL-positive apoptotic cells marginally increased in all mitomycin C groups whereas Ki67-positive mitotic cells decreased significantly following mitomycin C application. A greater …

Role Of KAtp Channels In Reduced Antinociceptive Effect Of Morphine In Streptozotocin-Induced Diabetic Mice, Vivek Sood, Ajay Sharma, Manjeet Singh

Pharmacy Faculty Articles and Research

The nociceptive effect was measured using withdrawal latency in tail flick test in mice rendered diabetic by administering streptozotocin (200 mg/kg, i.p.). The antinociceptive effect of morphine (4 and 8 mg/kg, s.c.) and cromakalim, a KATP channel opener, (0.3, 1 and 2 micrograms, i.c.v.) was significantly reduced in diabetic mice. Moreover, co-administration of cromakalim(0.3 microgram) did not alter the reduced antinociceptive effect of morphine(4 mg/kg) in diabetic mice. Spleenectomy in diabetic mice restored the decrease in antinociceptive effect of morphine and cromakalim. Multiple dose treatment with insulin to maintain euglycaemia for 3 days in diabetic mice prevented the decrease in …

Effect Of Actinomycin D And Cycloheximide On Ischemic Preconditioning-Induced Delayed Cardioprotective Effect In Rats, Devinder Singh, Ajay Sharma, Manjeet Singh

Pharmacy Faculty Articles and Research

The present study was designed to investigate the effect of actinomycin D, a transcription inhibitor, and cycloheximide, a translation inhibitor, on the delayed cardioprotective effect of ischemic preconditioning. Left thoracotomy was performed in anaesthetized rats at 4th/5th intercostal space and polypropylene suture (5-0) was employed to occlude left common coronary artery. Ischemic preconditioning was produced by four episodes of 5 min of coronary artery occlusion followed by 5 min of reperfusion and thoracic cavity was sutured. Left thoracotomy was performed again after 24 hr of ischemic preconditioning and left coronary artery was occluded for 30 min followed by reperfusion for …