Organisms Commons^™

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 …

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 …

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 …

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 …

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 …

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 …

Liquid Chromatography-Tandem Mass Spectrometry For The Determination Of Methylprednisolone In Rat Plasma And Liver After Intravenous Administration Of Its Liver-Targeted Dextran Prodrug, Shuang-Qing Zhang, Helen R. Thorsheim, Suman Penugonda, Venkateswaran C. Pillai, Quentin R. Smith, Reza Mehvar

Pharmacy Faculty Articles and Research

A specific and sensitive liquid chromatography (LC)-tandem mass spectrometric method for quantitative determination of methylprednisolone (MP) in rat plasma and liver was developed and validated using triamcinolone acetonide as an internal standard. Liquid-liquid extraction using tert-butyl methyl ether was used to extract the drug and the internal standard from plasma and liver. The separation of MP was performed on a C(18) column with a mobile phase of acetonitrile:0.5% formic acid aqueous solution (85:15, v/v) over 4 min. The assay was based on the selected reaction monitoring transitions at m/z 375 -> 161 for MP in plasma, 375 -> 357 for …

Helical Peptides Derived From Lactoferrin Bind Hepatitis C Virus Envelope Protein E2, Reem Beleid, Donna Douglas, Norman Kneteman, Kamaljit Kaur

Pharmacy Faculty Articles and Research

Hepatitis C virus is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. Hepatitis C virus envelope 2 glycoprotein (E2) binds several cell‐surface molecules that act as receptor candidates mediating hepatitis C virus entry into hepatocytes. Peptides derived from human lactoferrin have been shown to bind hepatitis C virus‐E2 protein thereby preventing hepatitis C virus entry in cultured hepatocytes. In this study, starting from a 33‐residue human lactoferrin‐derived peptide, a number of biotin‐linked α‐peptides were synthesized and investigated for their E2 protein binding activity. E2 protein from hepatitis C virus genotype 1b …

Effects Of Methylprednisolone And Its Liver-Targeted Dextran Prodrug On Ischemia–Reperfusion Injury In A Rat Liver Transplantation Model, Anjaneya Chimalakonda, Reza Mehvar

Pharmacy Faculty Articles and Research

Purpose. To evaluate the effectiveness of a liver-targeted dextran prodrug (DMP) of methylprednisolone ( MP) in cold preservation-warm reperfusion injury associated with liver transplantation.

Methods. The effects of donor pretreatment with single 5 mg/kg doses of MP or DMP on ischemia reperfusion damage to the liver were studied after 8 or 24 h of cold preservation in both isolated perfused rat liver (IPRL) and syngeneic orthotopic rat liver transplantation (OLT) models.

Results. In IPRL studies, donor pretreatment with DMP, and to a lesser degree MP, significantly improved the uptake of hyaluronic acid (HA), a marker of endothelial …

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 …

Attenuation Of Acute Rejection In A Rat Liver Transplantation Model By A Liver-Targeted Dextran Prodrug Of Methylprednisolone, Anjaneya Chimalakonda, Donald L. Montgomery, Jon A. Weidanz, Imam H. Shaik, Justin H. Nguyen, John J. Lemasters, Eiji Kobayashi, Reza Mehvar

Pharmacy Faculty Articles and Research

Background. The use of methylprednisolone (MP) and other corticosteroids for the treatment of acute liver allograft rejection is associated with severe toxicities in nontarget tissues. Therefore, selective delivery of NIP to the liver may improve its efficacy and alleviate its side effects. We investigated the effects of a novel liver-targeted dextran prodrug of MP (DMP) in an orthotopic rat liver transplantation (OLT) model.

Methods. The model consisted of a high responder rejection strain combination (Dark Agouti donors and Lewis recipients). Liver recipients were intravenously administered saline or a single subtherapeutic dose of MP (5 mg/kg) as the parent …

Hepatic Disposition Of Cyclosporine A In Isolated Perfused Rat Livers, Reza Mehvar, Anjaneya Chimalakonda

Pharmacy Faculty Articles and Research

PURPOSE. To develop an isolated perfused rat liver model to study the hepatic disposition of cyclosporine A (CyA) in both sexes.

METHODS. Livers were isolated from male (n = 6) and female (n = 7) rats and perfused with a physiological buffer in a single-pass manner. A bolus 1-mg dose of CyA was injected into the inlet catheter and periodical samples (0-15 min) were collected from the outlet perfusate. The concentrations of CyA in the outlet perfusate, collected bile (0-15 min), and liver tissue (at the end of perfusion) were quantitated by HPLC and subjected to statistical moment analysis. …

Dextran-Methylprednisolone Succinate As A Prodrug Of Methylprednisolone: Plasma And Tissue Disposition, Xiaoping Zhang, Reza Mehvar

Pharmacy Faculty Articles and Research

Plasma and tissue disposition of a macromolecular prodrug of methylprednisolone (MP), dextran (70 kDa)–methylprednisolone succinate (DMP), was studied in rats. Single 5‐mg/kg doses of DMP or unconjugated MP were administered into the tail veins of different groups of rats (n  = 4/group/time point). Blood (cardiac puncture) and tissues (liver, spleen, kidney, heart, lung, thymus, and brain) were collected at various times after DMP (0–96 h) or MP (0–2 h) injections. Concentrations of DMP and MP in samples were analyzed by size‐exclusion chromatography (SEC) and reversed‐phase high‐performance liquid chromatography (HPLC), respectively. Conjugation of MP with 70‐kDa dextran resulted in 22‐, …

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 …

Effect Of Inhibitors Of Arachidonic Acid Metabolism On Corneal Neovascularization In The Rat, W. L. Haynes, A. D. Proia, G. K. Klintworth

Osteopathic Medicine, Jerry M. Wallace School of

No abstract provided.