Musculoskeletal, Neural, and Ocular Physiology Commons^™

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 …

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 …

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 …

"Transduction Efficiency Of Aav 2/6, 2/8 And 2/9 Vectors For Delivering Genes In Human Corneal Fibroblasts ", Ajay Sharma, Arkasubhra Ghosh, Eric T. Hansen, Jason M. Newman, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

In the present study, cellular tropism and relative transduction efficiency of AAV2/6, AAV2/8 and AAV2/9 vectors have been tested for the cornea using primary cultures of human corneal fibroblasts. The AAV6, AAV8 and AAV9 serotypes having AAV2 ITR plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Primary cultures of human corneal fibroblasts were exposed to AAV infectious particles at two different doses (1×105 and 2×105 MOI). Cytochemistry and enzyme assays were used to measure delivered transgene expression in samples collected at 4 and 30 hours after AAV infection …

Role Of Transforming Growth Factor Beta In Corneal Function, Biology And Pathology, Ashish Tandon, Jonathan C. K. Tovey, Ajay Sharma, Rangan Gupta, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

Transforming growth factor-beta (TGFβ) is a pleiotropic multifunctional cytokine that regulates several essential cellular processes in many parts of the body including the cornea. Three isoforms of TGFβ are known in mammals and the human cornea expresses all of them. TGFβ1 has been shown to play a central role in scar formation in adult corneas whereas TGFβ2 and TGFβ3 have been implicated to play a critical role in corneal development and scarless wound healing during embryogenesis. The biological effects of TGFβ in the cornea have been shown to follow SMAD dependent as well as SMAD-independent signaling pathways depending upon cellular …

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 …

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 …

Altered Regulation Of Contraction-Induced Akt/Mtor/P70s6k Pathway Signaling In Skeletal Muscle Of The Obese Zucker Rat, Anjaiah Katta, Sunil K. Kakarla, Miaozong Wu, Satyanarayana Paturi, Murali K. Gadde, Ravi K. Arvapalli, Madhukar Babu Kolli, Kevin M. Rice, Eric R. Blough

MIIR Faculty Research

Increased muscle loading results in the phosphorylation of the 70 kDa ribosomal S6 kinase (p70S6k), and this event is strongly correlated with the degree of muscle adaptation following resistance exercise. Whether insulin resistance or the comorbidities associated with this disorder may affect the ability of skeletal muscle to activate p70S6k signaling following an exercise stimulus remains unclear. Here, we compare the contraction-induced activation of p70S6k signaling in the plantaris muscles of lean and insulin resistant obese Zucker rats following a single bout of increased contractile loading. Compared to lean animals, the basal phosphorylation of p70S6k (Thr389; 37.2% and Thr421/Ser424; 101.4%), …

Performance Of A Hip Protector Depends On Its Position During A Fall, Woochol J. Choi, Joaquin A. Hoffer, Stephen N. Robinovitch

Physical Therapy Faculty Articles and Research

"Hip protectors are designed to attenuate and redistribute the force applied to the hip region during a fall, and thereby reduce risk for hip fracture [1]. However, little information exists on the effectiveness of hip protectors in achieving these goals, and how this is altered by displacement of the hip protector relative to the greater trochanter (GT). In the current study, we tested these issues."

Aging-Associated Dysfunction Of Akt/Protein Kinase B: S-Nitrosylation And Acetaminophen Intervention, Miaozong Wu, Anjaiah Katta, Murali K. Gadde, Hua Liu, Sunil K. Kakarla, Jacqueline Fannin, Satyanarayana Paturi, Ravi K. Arvapalli, Kevin M. Rice, Yeling Wang, Eric R. Blough

MIIR Faculty Research

Background: Aged skeletal muscle is characterized by an increased incidence of metabolic and functional disorders, which if allowed to proceed unchecked can lead to increased morbidity and mortality. The mechanism(s) underlying the development of these disorders in aging skeletal muscle are not well understood. Protein kinase B (Akt/PKB) is an important regulator of cellular metabolism and survival, but it is unclear if aged muscle exhibits alterations in Akt function. Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention.

Principal Findings: Compared to 6- and 27-month rats, …

Effects Of Estrogen On Muscle Damage In Response To An Acute Resistance Exercise Protocol, Megan R. Wolf

Honors Scholar Theses

Creatine Kinase (CK) is used as a measure of exercise-induced muscle membrane damage. During acute eccentric (muscle lengthening) exercise, muscle sarcolemma, sarcoplasmic reticulum, and Z-lines are damaged, thus causing muscle proteins and enzymes to leak into the interstitial fluid.

Strenuous eccentric exercise produces an elevation of oxygen free radicals, which further increases muscle damage. Muscle soreness and fatigue can be attributed to this membrane damage. Estradiol, however, may preserve membrane stability post-exercise (Brancaccio, Maffulli, & Limongelli, 2007; Carter, Dobridge, & Hackney, 2001; Tiidus, 2001). Because estradiol has a similar structure to Vitamin E, which is known to have antioxidant properties, …

Lean And Obese Zucker Rats Exhibit Different Patterns Of P70s6 Kinase Regulation In The Tibialis Anterior Muscle In Response To High-Force Muscle Contraction, Anjaiah Katta, Sunil K. Karkala, Miaozong Wu, Sarath Meduru, Devashish H. Desai, Kevin M. Rice, Eric R. Blough

MIIR Faculty Research

Increased phosphorylation of the 70-kDa ribosomal S6 kinase (p70S6k) signaling is strongly correlated with the degree of muscle adaptation following exercise. Herein we compare the phosphorylation of p70S6k, Akt, and mammalian target of rapamycin (mTOR) in the tibialis anterior (TA) muscles of lean and obese Zucker rats following a bout of eccentric exercise. Exercise increased p70S6k (Thr389) phosphorylation immediately after (33.3 ± 7.2%) and during [1 h (24.0 ± 14.9%) and 3 h (24.6 ± 11.3%)] recovery in the lean TA and at 3 h (33.5 ± 8.0%) in the obese TA Zucker rats. mTOR (Ser2448) phosphorylation was elevated in …

Regulation Of Alpha- And Beta-Actin Isoforms In The Contracting A7r5 Smooth Muscle Cell, Dawn Leah Brown-Turner

Theses, Dissertations and Capstones

Two isoforms of actin have been found to be present in A7r5 smooth muscle cells, α- and β-actin. This body of work sets out to examine the different regulatory factors of the two actin isoforms during smooth muscle contraction. The response of the actin isoforms to phorbol 12, 13-dibutyrate (PDBu) is markedly different. α-Actin remodels to podosomes around the periphery of the cell, while β-actin merely shortens. One protein involved in smooth muscle contraction induced by PDBu is protein kinase C (PKC). Two inhibitors of PKC, staurosporine and bisindolymaleimide, were used prior to PDBu stimulation and after PDBu stimulation to …

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 …

Three-Dimensional Arm Movements At Constant Equi-Affine Speed, Frank E. Pollick, Uri Maoz, Amir A. Handzel, Peter J. Giblin, Guillermo Sapiro, Tamar Flash

Psychology Faculty Articles and Research

It has long been acknowledged that planar hand drawing movements conform to a relationship between movement speed and shape, such that movement speed is inversely proportional to the curvature to the power of one-third. Previous literature has detailed potential explanations for the power-law’s existence as well as systematic deviations from it. However, the case of speed-shape relations for three-dimensional (3D) drawing movements has remained largely unstudied. In this paper we first derive a generalization of the planar power law to 3D movements, which is based on the principle that this power law implies motion at constant equi-affine speed. This generalization …

Effects Of Aging And Gender On Regulators Of Muscle Adaptation In F344/Bn Rat Model, Satyanarayana Paturi

Theses, Dissertations and Capstones

Sarcopenia is the loss of muscle mass and strength that occurs with aging. Here we examine the effects of aging and gender on the regulation of molecules believed to regulate muscle growth and adaptation in the F344/BN rat. In male animals, soleus and EDL muscle/body weight ratio declined continuously with aging while muscle atrophy in female animals plateaued at 26-months and remained constant thereafter. Aging increased the phosphorylation of protein kinase-B (Akt) and the mammalian target of rapamycin (mTOR) in the female but not male soleus muscle. This finding was associated with the attenuation of muscle atrophy observed in female …

Impaired Overload-Induced Hypertrophy In Obese Zucker Rat Slow-Twitch Skeletal Muscle, Anil Kumar Gutta

Theses, Dissertations and Capstones

How insulin resistant muscle responds to mechanical overload (MOV) is not well understood. Using immunohistochemical analysis and immunoblotting, we investigated whether Lean Zucker (LZ) and insulin resistant Obese Zucker rats (OZ) respond in a similar fashion to MOV. Five young adult (2 months old) male LZ and OZ rats were subjected to a surgical ablation of the gastrocnemius muscle and overloaded for 8 weeks. MOV-induced increases in soleus muscle mass and average fiber cross-sectional area were attenuated in OZ compared to LZ animals. This reduction in OZ muscle adaptation was associated with decreased activity / phosphorylation levels of STAT3, β-catenin …

Pdgf-Driven Proliferation, Migration, And Il8 Chemokine Secretion In Human Corneal Fibroblasts Involve Jak2-Stat3 Signaling Pathway, Neeraj Vij, Ajay Sharma, Mahesh Thakkar, Sunilima Sinha, Rajiv R. Mohan

Pharmacy Faculty Articles and Research

Purpose: Platelet-derived growth factor (PDGF) is associated with corneal fibroblast migration and proliferation and plays an important role in corneal wound healing. However, the intracellular mechanisms of PDGF-mediated functions in corneal fibroblasts are poorly understood. We tested the hypothesis that PDGF functional activities in the cornea involve the Janus kinase-2/signal transducers and activators of transcription-3 (JAK2-STAT3) signaling pathway and whether PDGF induces the expression of suppressors of cytokine signaling 3 (SOCS3), belonging to the novel family of feedback regulators of cytokine and growth factor activities.

Methods: Human corneal fibroblast (HSF) cultures were used as an in vitro model for functional …

The Neural Correlates Of Non-Spatial Working Memory In Velocardiofacial Syndrome (22q11.2 Deletion Syndrome), Wendy R. Kates, Beth R. Krauss, Nuria Abdulsabur, Dierdre Colgan, Kevin M. Antshel, Anne Marie Higgins, Robert J. Shprintzen

Communication Disorders Faculty Publications

Velocardiofacial syndrome (VCFS), also known as 22q11.2 deletion syndrome, is a neurogenetic disorder that is associated with both learning disabilities and a consistent neuropsychological phenotype, including deficits in executive function, visuospatial perception, and working memory. Anatomic imaging studies have identified significant volumetric reductions in the parietal lobe of individuals with VCFS, but several studies have reported that the frontal lobe is relatively preserved. We used functional magnetic resonance imaging to investigate the neural correlates of non-spatial working memory in 17 youths with VCFS, 10 of their unaffected siblings, and 10 community controls (with the same proportion of learning disabilities as …

The Influence Of Ambient Lighting Levels On Postural Sway In Healthy Children Ages 9 To 11, Yvette Blanchard, Rebecca Mcveigh, Megan Graham, Melissa Cadet, Kabulo Mwilambwe, Christi Scott

All PTHMS Faculty Publications

The purpose of this study was to determine whether postural sway in healthy children varied in different levels of ambient lighting. Twelve boys and 26 girls with a mean age of 118 months stood on a force platform under three conditions: eyes closed, eyes opened in regular light (200 lx) and eyes opened in dim light (3 lx). Analysis of variance and pairwise comparisons revealed significantly more postural sway with the eyes closed condition compared to the regular and dim light conditions but no differences between the regular and dim light conditions. While our results on postural sway during the …

Mlck/Actin Interaction In The Contracting A7r5 Cell And Vascular Smooth Muscle, Sean Eric Thatcher

Theses, Dissertations and Capstones

Myosin light chain kinase (MLCK) is an enzyme that phosphorylates the serine-19 residue on myosin regulatory light chains (MLCs) which serves to activate the Mg2+-ATPase of myosin. This catalytic activity is thought to be the primary role of MLCK; however, it has recently been suggested that MLCK’s actin binding and bundling properties may also be of importance in smooth muscle contraction. In the absence of calcium and calmodulin (CaM), MLCK will bundle actin filaments with its N-terminus. During calcium influx and subsequent CaM activation, MLCK binding to actin decreases resulting in unbundling of actin filaments and allows myosin and actin …

Shear Stress Induced Mtor Signaling In Cultured A7r5 Aortic Smooth Muscle Cell Line, Sriram Prasad Mupparaju

Theses, Dissertations and Capstones

Vascular intervention procedures such as balloon angioplasty and stent implantation lead to denudation of endothelium exposing the underlying smooth muscle layer to effects of blood shear stress. The effect of shear stress on smooth muscle cell signaling has not been studied in depth. In this study, we examined time dependent changes in activation of Akt, mTOR and p70S6k related signaling in response to orbital shear stress on cultured A7r5 cells. A7r5 cells cultured to subconfluence on 150mm culture dishes were subjected to orbital shear of 9.8dynes/cm 2 for zero, 5 min, 15 min, 30 min, 1h, 4h and 24h. The …

Association Of Smooth Muscle Myosin And Its Carboxyl Isoforms With Actin Isoforms In Aorta Smooth Muscle, Jason Edward Black

Theses, Dissertations and Capstones

The contraction mechanism of smooth muscle is not fully understood. The primary interaction that leads to the formation of tension, the myosin-actin crossbridge, has been studied extensively. However, even this aspect of the contraction has proven not to be as simple as it might seem. There are several isoforms of smooth muscle myosin and actin, and the differences in the activities of these isoforms and their interactions during the contractile process are largely unknown. The studies to be discussed are directed at the determination of the interaction of these isoforms during the contraction of rat aortic smooth muscle. Chapter II …

Lean And Obese Zucker Rats Exhibit Different Patterns Of P70s6kinase Regulation In The Tibialis Anterior Muscle In Response To High Force Muscle Contraction, Anjaiah Katta

Theses, Dissertations and Capstones

Increased muscle loading results in phosphorylation of the 70 kDa ribosomal S6 kinase (p70s6k) signaling pathway and this event strongly correlates with degree of muscle adaptation following resistance exercise. Here, we compared the basal and contraction- induced phosphorylation of p70s6k, Akt and mTOR in tibialis anterior muscles of lean and obese Zucker rats. Immunoblotting demonstrated differences in level of basal p70S6k phosphorylation (Thr 389) in the normal and diabetic TA. HFES had an increase in p70S6k (Thr389) phosphorylation at 0-, 1- and 3-hr in lean TA and only at 3-hr in obese TA. mTOR (Ser 2448) Phosphorylation was elevated in …

Age-Related Dystrophin–Glycoprotein Complex Structure And Function In The Rat Extensor Digitorum Longus And Soleus Muscle, Kevin M. Rice, Deborah L. Preston, David Neff, Michael L. Norton, Eric R. Blough

Eric Blough

Age-Related Dystrophin–Glycoprotein Complex Structure And Function In The Rat Extensor Digitorum Longus And Soleus Muscle, Kevin M. Rice, Deborah L. Preston, David Neff, Michael L. Norton, Eric R. Blough

Kevin M Rice

Age-Related Dystrophin–Glycoprotein Complex Structure And Function In The Rat Extensor Digitorum Longus And Soleus Muscle, Kevin M. Rice, Deborah L. Preston, David Neff, Michael L. Norton, Eric R. Blough

Deborah L Preston

Abdominal Pain With Rigidity Secondary To The Anti-Emetic Drug Metoclopramide, Nadeem Ullah Khan, Junaid A. Razzak

Department of Emergency Medicine

We report a case of abdominal pain with rigidity, mimicking an acute abdomen, caused by metoclopramide, a common anti-emetic drug. Extrapyramidal symptoms are commonly reported side-effects of this medication. They generally include involuntary movements of limbs, torticollis, oculogyric crisis, rhythmic protrusion of tongue, trismus, or dystonic reactions resembling tetanus, etc. Abdominal rigidity due to this medication, resembling an acute abdomen, has not been reported previously. This case report illustrates the importance of considering medication side-effects when evaluating a patient with abdominal pain and rigidity.

Diverse Roles Of Pkcα In Vascular Smooth Muscle Contraction, Ava Caudill Dykes

Theses, Dissertations and Capstones

A complete understanding of the mechanisms underlying smooth muscle contractility has proven elusive due to the many interacting factors thought to influence contractile behavior in this muscle type. One such factor, protein kinase Cα (PKCα), is proposed to play a central role in both the activation and modulation of smooth muscle contraction. PKCα is activated through a G-protein coupled receptor and is subsequently translocated to its site of action. Although the mechanisms regulating PKCα site specific translocation are not completely understood, it is thought that this process is essential for PKCα specificity of action within the cell. In chapter II …