2006 merck/merial - School of Veterinary Medicine - Louisiana State ...

transduction, cellular transformation and oncogenesis. Calcineurin (CaN) is an 80-kDa protein phosphatase, regulated byintracellular Ca2+ concentrations, that has been found to be involved in the proinflammatory response. The goal of this studywas to test the hypothesis that expression and activity of CaN and NMT is increased in pulmonary inflammation caused byinhalation of swine barn air or intratracheal instillation of E. coli lipopolysaccharide (LPS). In order to accomplish this, micewere either exposed to barn air for 1 day (n=4), LPS (n=5) or used as controls (n=6). Sections obtained from the lung tissueswere stained with hematoxylin and eosin or with antibodies against NMT and CaN. H&E staining revealed an increase ininflammation characterized by septal recruitment of neutrophils and macrophages along with dilatation of perivascular spacesin mice exposed to the LPS or the barn air compared to the controls.The degree of inflammatory changes appeared to be similar in the LPS and the barn groups. Immunohistochemistryrevealed CaN and NMT expression in macrophages in all the groups. However, there were no differences in the number ofcells positive for CaN and NMT among the groups (P>0.05). Currently, we are analyzing lung tissues for CaN and NMTactivity. The results from this study show that immunohistologic expression of CaN and NMT remains unaltered followingexposure to the LPS or the barn air.Pig coronary atherosclerosis and the effects of endurance exercise on mRNA expressionThandeka Ngwenyama*, Dr. JR Turk, Jennifer CasatiCollege of Veterinary Medicine, University of Missouri-ColumbiaAtherosclerosis and related illnesses are the leading cause of illness and death in the U.S. High blood cholesterol,obesity, and physical inactivity are predisposing risk factors for the early progression of cardiovascular disease in humans.Our hypothesis is that endurance exercise will decrease oxidized low-density lipoprotein (oxLDL) and increase endothelialnitric oxide synthase (eNOS) and superoxide dismutase (SOD1) in pig coronaries. Sexually mature (9-12) pigs are fed dailya high fat, high cholesterol diet (HFC) and assigned to either sedentary (Sed) or exercise (Ex) groups. At the end of 20weeks, the pigs are anesthetized and the chest opened to achieve euthanasia. Cross sections of harvested common coronaryarteries are frozen using liquid nitrogen and immunohistochemistry for oxLDL was performed to select cells according tophenotypic and functional characteristics. Laser capture microdissection (LCM) techniques allow extraction ofmorphologically distinct cells for molecular analysis. Pure cell populations of oxLDL –ve and +ve cells are extracted usingan infrared laser pulse to select and adhere discrete cells to a thermoplastic film. The extracted cells are then transferred intoa digestion buffer for extraction of mRNA and subsequent analysis using RT-PCR for eNOS and SOD1. The enzyme eNOScatalyzes the conversion of L-arginine to nitric oxide (NO) which facilitates vasodilation. SOD1 protects NO fromdegradation by scavenging the superoxide anion (O 2·). Endothelial dependent vasodilation is proposed to be enhanced byexercise training through the mechanism of increased expression of the gene coding for eNOS and/or prolong the biologicalhalf-life of NO by an upregulation of SOD1 expression. Gene expression analysis patterns in pathological processes such asatherosclerosis are useful for diagnostic, prognostic, preventative treatment and in the future could allow individualizedtreatments for patients with different disease stages.Analysis of retinal molecular marker expression associated with canine inherited cone degenerationDM Niculescu 1 *, GD Aguirre 2 , GM Acland 3 , AM Komáromy 2Auburn University, College of Veterinary Medicine, Auburn, AL 1 , Department of Clinical Studies, School ofVeterinary Medicine, University of Pennsylvania, Philadelphia, PA 2 , Baker Institute, Cornell University, Ithaca,NY 3Purpose: To evaluate expression of selected gene products in the retina of dogs with inherited cone degeneration(cd), a model for human achromatopsia.Methods: We performed immunohistochemical labeling on canine retinal tissues (cd affected and non-affected) atfive different ages between 4 weeks and 3.75 years. This time period covers normal photoreceptor development as well as theearly stages of cd. We used the following antibodies to label cone photoreceptors: red/green (L/M) opsin, blue (S) opsin,human cone arrestin (hcArr), and clusterin-like 1 protein (CLUL1). We labeled other retinal cells using antibodies againstRPE65, rhodopsin, synaptophysin, protein kinase C and Goa, calbindin D-28k, GABA, glial fibrillary acidic protein andglutamine synthetase. Antibodies for brain-derived neurotrophic factor and TrkB were also used.Results: Analysis of affected retinal tissues revealed that L/M and S cones were present throughout the time periodcovered. However, the observed L/M and S opsin expression patterns demonstrated an age-related degenerative processcharacterized by decreasing cone numbers and altered cone morphology. CLUL1 labeling of the cone outer segments (OS)did not appear different between the affected and non-affected tissues. Arr was detected in the OS but not inner segments (IS)of most affected cones; in contrast, both OS and IS of non-affected cones were labeled. In the non-affected tissues TrkB wasexpressed in retinal ganglion cells and cone IS; labeling intensity appeared to increase with age. There was no TrkB labelingof cone IS in the affected retinas. We did not detect changes in the molecular markers of the retinal pigment epithelium, rods,and cells of the inner retina.Conclusions: Cones were present at all disease stages studied, which may prove useful for future rescue attempts. Incontrast to non-affected retinas, TrkB was not expressed in cones of affected retinas and Arr could not be found in most coneIS.105