VTechWorks Repository :: Browsing by Author "Pleasant, R. Scott"

Browsing by Author "Pleasant, R. Scott"

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Biochemical Characterization of Normal Navicular Bone Flexor Surface Cartilage
Vits, Lucia Carolina (Virginia Tech, 2002-11-12)
Cartilage tissue specimens were obtained from the flexor surface of the navicular bone and distal radiocarpal bone articular surface (controls) from 8 horses 2 to 5 years old. Water, DNA, total collagen, total glycosaminoglycans, chondroitin sulphate, and keratan sulphate contents were determined. The results from each site were compared and the differences were analyzed by paired t-test (P < 0.05). Significant differences were determined between the water content of the navicular bone flexor surface cartilage (68.32± 3.46 % ) and the distal radiocarpal bone articular surface cartilage (60.60± 4.09%). The total DNA content, total glycosaminoglycan content, total chondroitin sulphate content, and total keratan sulphate for the flexor surface of the navicular bone was: 524.51± 92.89 ng, 0.1533± 0.0338 mg, 0.1018± 0.0197 mg 0.0800± 0.0176 mg, and 0.0092± 0.0037 mg per mg of dry weight cartilage, respectively. The total DNA content, total glycosaminoglycan content, total chondroitin sulphate content, and total keratan sulphate for the distal radiocarpal articular surface cartilage was: 508.80± 70.16 ng, 0.1686± 0.00838 mg, 0.0919± 0.0191, 0.0615± 0.0109 mg, and 0.0074± 0.0029 mg per mg dry weight cartilage, respectively. Not significant differences were determined between these values. We concluded that the cartilage of the flexor surface of the navicular bone is biochemically similar to hyaline articular cartilage, but differs from previous descriptions of fibrocartilage. Further studies are needed to determine types and proportions of collagen types of the flexor surface of the normal navicular bone. These findings establish a basis of comparison to assess navicular cartilage in aging, disease, and repair.
Can levamisole upregulate the equine cell mediated immune response in vitro?
Santonastaso, Amy Marie (Virginia Tech, 2016-07-19)
Equine Protozoal Myeloencephalitis (EPM) is arguably the most common and costly equine neurologic diseases nationwide. The national seroprevalence is >50%, but only 0.5-1% of all horses develops disease during their lifetimes. Some EPM affected horses have decreased immune response. A cell-mediated immune response has been shown to be protective for development of EPM after infection with Sarcocystis neurona in mouse models. Levamisole has been proposed as an adjunctive therapy for EPM to upregulate the cell-mediated immune response based on positive results in other species, but there are very limited studies in equids. We hypothesized that levamisole will upregulate the equine cell-mediated macrophage (M1) dendritic cell (DC1) CD4 T-helper 1 (Th1) CD8 Tc1 immune response in vitro. The first aim was to determine optimal conditions and effects of levamisole on cellular proliferation. Equine PBMCs were harvested from ten horses seronegative for S. neurona. The cells were cultured alone, or with one of the mitogens: concanavalin A (ConA) or phorbol 12-myristate 13-acetate and ionomycin (PMA/I), or with a combination of the above mitogens and levamisole at several conditions. Cellular proliferation was assessed using a colorimetric bromodeoxyuridine ELISA assay. The second aim was to determine the ability of levamisole, under optimized conditions, to upregulate the M1 DC1 CD4Th1 CD8 Tc1 response in vitro based on activation and function. PBMCs from the same 10 horses were cultured with each of the following: no stimulation, conA, and levamisole with and without ConA. To determine proliferation of each specific subset, cells were labeled with a fluorescent dye, CellTrace. Proliferation was determined based on dye dilution using flow cytometry. To determine the effects of levamisole on the specific immune response, cell subsets were labeled with fluorescent antibodies for cell surface markers (CD4, CD8, CD21, CD172a, CD14) and dendritic and macrophage activations markers (MHC Class II, CD86). Induction of T-regs was based on FoxP3 expression. Immune phenotypes were determined based on intracellular cytokine expression (IFNɣ, IL4, IL10). Study results indicate that levamisole alone did not significantly alter PBMC proliferation compared to the response of unstimulated cells. Cells cultured with either ConA or PMA/I resulted in a statistically significant increase (P<0.05) in proliferation compared to unstimulated cells. Cells cultured with ConA and levamisole at 1µg/mL resulted in a significant decrease (P<0.05) in proliferation compared with cells cultured with ConA alone. Flow cytometry data failed to elucidate the specific immune phenotype that is affected by levamisole. Subjectively, there appeared to be a trend for inceased IFNɣ production by CD14 and CD172a positive cells (macrophages and dendritic cells) and a decrease in IFNɣ production by CD4 and CD8 positive cells (T-lymphocytes). These results demonstrate that levamisole downregulates ConA stimulated PBMC proliferation. Based on these in vitro results, further studies to determine the effectiveness of levamisole on modulating the equine immune system in vivo and to more specifically evaluate the immune cell subets affected by levamisole are warranted.
Can levamisole upregulate the equine cell-mediated macrophage (M1) dendritic cell (DC1) T-helper 1 (CD4 Th1) T-cytotoxic (CD8) immune response in vitro?
Witonsky, Sharon G.; Buechner-Maxwell, Virginia A.; Santonastasto, Amy; Pleasant, R. Scott; Werre, Stephen R.; Wagner, Bettina; Ellison, Siobhan; Lindsay, David S. (Wiley, 2019-03-01)
Background: Equine protozoal myeloencephalitis (EPM) is a common and devastating neurologic disease of horses in the United States. Because some EPM-affected horses have decreased immune responses, immunomodulators such as levamisole have been proposed as supplemental treatments. However, little is known about levamisole's effects or its mechanism of action in horses. Objective: Levamisole in combination with another mitogen will stimulate a macrophage 1 (M1), dendritic cell 1 (DC1), T-helper 1 (CD4 Th1), and T-cytotoxic (CD8) immune response in equine peripheral blood mononuclear cells (PBMCs) in vitro as compared to mitogen alone. Animals: Ten neurologically normal adult horses serologically negative for Sarcocystis neurona. Methods: Prospective study. Optimal conditions for levamisole were determined based on cellular proliferation. Peripheral blood mononuclear cells were then cultured using optimal conditions of mitogen and levamisole to identify the immune phenotype, based on subset-specific activation markers, intracellular cytokine production, and cytokine concentrations in cell supernatants. Subset-specific proliferation was determined using a vital stain. Results: Concanavalin A (conA) with levamisole, but not levamisole alone, resulted in a significant decrease (P <.05) in PBMC proliferation compared to conA alone. Levamisole alone did not elicit a specific immune phenotype different than that induced by conA. Conclusion and Clinical Importance: Levamisole co-cultured with conA significantly attenuated the PBMC proliferative response as compared with conA. If the mechanisms by which levamisole modulates the immune phenotype can be further defined, levamisole may have potential use in the treatment of inflammatory diseases.
Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing
Parkinson, Nicholas J.; Buechner-Maxwell, Virginia A.; Witonsky, Sharon G.; Pleasant, R. Scott; Werre, Stephen R.; Ahmed, Sattar Ansar (PLOS, 2017-05-26)
The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces differential microRNA expression in equine peripheral blood mononuclear cells in a manner comparable to humans. Illumina Next Generation Sequencing was used to characterize the basal microRNA transcriptome in isolated peripheral blood mononuclear cells from healthy adult horses, and to evaluate LPS-induced changes in microRNA expression in cells cultured for up to four hours. Selected expression changes were validated using quantitative reverse-transcriptase PCR. Only miR-155 was significantly upregulated by LPS, changing in parallel with supernatant tumor necrosis factor-α concentration. Eight additional microRNAs, including miR-146a and miR-146b, showed significant expression change with time in culture without a clear LPS effect. Target predictions indicated a number of potential immunity-associated targets for miR-155 in the horse, including SOCS1, TAB2 and elements of the PI3K signaling pathway, suggesting that it is likely to influence the acute inflammatory response to LPS. Gene alignment showed extensive conservation of the miR-155 precursor gene and associated promoter regions between horses and humans. The basal and LPS-stimulated microRNA expression pattern characterized here were similar to those described in human leukocytes. As well as providing a resource for further research into the roles of microRNAs in immune responses in horses, this will facilitate inter-species comparative study of the role of microRNAs in the inflammatory cascade during endotoxemia and sepsis.
The Comparison of Airway Responses of Normal Horses Fed Round Bale versus Square Bale Hay
Larson, Jennifer Lynn (Virginia Tech, 2012-06-13)
Background – Feeding horses round bale hay (RBH) has been associated with airway inflammation. The purpose of this study was to determine if horses fed RBH for a 6-week period demonstrated more evidence of airway inflammation than horses fed square bale hay (SBH) of comparable quality. Hypothesis - The respiratory health of horses fed RBH will not differ from horses fed SBH of comparable quality. Animals – Two feeding groups of 15 healthy horses (mixed ages, breeds) from the University riding program. Methods – This was a prospective study performed during fall of 2009. At the beginning and end of a 6- week feeding trial, horses were examined (physical, upper airway endoscopic) and samples (tracheal aspirate (TA), bronchoalveolar lavage (BAL)) collected for cytology and/or bacterial/fungal culture. Hay was analyzed for nutritional value and bacterial/fungal content. Results – Horses fed RBH demonstrated an increase in pharyngeal lymphoid hyperplasia (p=0.0143) and percentage neutrophils (p=0.0078) in the TA samples post-feeding as compared to pre-feeding values. Nutritional analysis of hay and measurements of bacterial/fungal load did not differ over time and/or between hay types. Conclusions and clinical importance – The identification of airway inflammation in the horses fed RBH indicates that factors associated with the manner in which the hay is fed and consumed contribute to the development of subclinical airway inflammation. RBH affords horses continuous daily exposure to hay and as horses bury their muzzles in the bale, exposure to particulate matter is likely increased. These factors may partially explain the response in horses fed RBH. Further studies are required to confirm these predictions.
The Effect of Growth Hormone on Pig Embryo Development in Vitro and an Evaluation of Sperm-Mediated Gene Transfer in the Pig
Bolling, Laura Clayton (Virginia Tech, 2001-08-20)
The objective of part one of this study was to determine if the presence of porcine growth hormone (pGH) during oocycte in vitro maturation (IVM) affected subsequent embryo development. Pig cumulus-oocyte complexes (COC) (n=987) were aspirated from slaughterhouse derived ovaries and cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10% v/v), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG, 10 IU/ml each), 10 ng/ml EGF, and with or without pGH (100 ng/ml) for 22 h. The COC were then cultured in the same medium with or without 100 ng/ml pGH, but without hormonal supplements for an additional 22 h. After the completion of maturation culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed spermatozoa for 8 h. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. Embryo development was assessed on d 6 of culture. The treatment groups were as follows: treatment 1 = control group cultured in IVM medium alone; treatment 2 = 100 ng/ml pGH present of the first 22 h of maturation culture and absent for the second 22 h of maturation culture; treatment 3 = 100 ng/ml pGH absent for the first 22 h of maturation culture, but present for the second 22 h of maturation culture; and treatment 4 = 100 ng/ml pGH present throughout the entire IVM period. Embryos were visually scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4- to 8-cell embryo, 4 = 9- to 16-cell embryo, 5 = morula, and 6 = blastocyst. The addition of pGH did not affect porcine embryo development as compared to the control (1.57 ± .08, 1.67 ± .08, 1.47 ± .08, and 1.60 ± .08, respectively; P > .10). Replicates within the study differed significantly from each other (P < .01) primarily because the development in replicate 6 was greater than for all others. There was a significant treatment by replicate interaction (P < .05); pGH added during the first 22 h of IVM and pGH added during the second 22 h of IVM in replicate 6 resulted in higher development scores than for controls and continuous pGH addition. However, in replicate 2, continuous pGH resulted in the greatest development. These results suggest that pGH may exert a stimulatory effect on embryo development when present in the IVM media; however, further studies using pGH in IVM culture are necessary. The objectives of the second part of the study were to examine aspects of intracytoplasmic sperm injection (ICSI) using membrane-disrupted spermatozoa, in vitro fertilization (IVF), and sperm-mediated gene transfer in the pig. Porcine oocytes were shipped overnight in maturation media at 39°C in a portable incubator. After 22 h of maturation culture, oocytes were washed in maturation medium without gonadotropins and cultured for an additional 22 h. Cumulus cells were removed and oocytes were divided into four treatment groups: treatment 1 = ICSI using membrane-damaged spermatozoa coincubated with linear green fluorescent protein (GFP) DNA; treatment 2 = ICSI using membrane damaged spermatozoa; treatment 3 = IVF with frozen-thawed spermatozoa coincubated with linear GFP DNA prior to IVF; treatment 4 = IVF with frozen-thawed spermatozoa with no DNA coincubation. Embryos were scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4-cell embryo, 4 = 5- to 8-cell embryo, 5 = 9- to 16-cell embryo, 6 = morula, and 7 = blastocyst. Although no overall difference in development score was observed following the four different treatments, a treatment difference among cleaved oocytes was observed when comparing only the two ICSI treatments (P < .05); development scores were greater in the ICSI treatment in which sperm were not coincubated with linear GFP DNA prior to injection than when the coincubation was performed (3.76 ± .21 vs. 3.13 ± .17, respectively). No differences in development score were observed in the two IVF treatments. The percentage of embryos expressing the GFP transgene on d 6 of culture following fertilization was 7.3% in the ICSI+GFP group and 0% in all other treatment groups. Thus, sperm-mediated gene transfer using ICSI in the pig has been demonstrated, although success rates were low.
Effects of Three Corticosteroids on Equine Articular Cocultures In Vitro
Trahan, Richard Angellas (Virginia Tech, 2018-06-08)
The objective was to compare the effects of three corticosteroids at various equimolar concentrations on equine articular explant co-cultures in an inflammatory environment. Synovial and osteochondral explant co-cultures from 6 equine cadavers were exposed to IL-1β (10 ng/mL) and various concentrations (10-4, 10-7, or 10-10 M) of MPA, TA, IPA. Concentrations of PGE2, MMP-13, LDH, and GAG in media were determined at 48 and 96 hours. Results indicated wells with low concentrations of MPA (10-7 and 10-10 M at 48 and 96 hours), TA (10-7 M at 48 hours and 10-7 and 10-10 M at 48 and 96 hours), and IPA (10-10 M at 48 hours) had significantly less PGE2 than positive control samples. Groups with low concentrations (10-7 and 10-10 M) of MPA and TA had significantly less PGE2 than the highest concentration (10-4 M) at 48 hours. Significantly less MMP-13 was detected for all concentrations of MPA, TA, and IPA at 96 hours. The LDH assay results indicated cytotoxicity only for samples treated with IPA at 10-4 M at 48 and 96 hours. GAG was significantly lower for samples treated with TA 10-7 M at 48 hours and MPA 10-10 M at 96 hours versus positive controls. These findings suggest corticosteroids at low concentrations mitigated the inflammatory and catabolic effects of IL-1β to a greater extent than high concentrations. Effects of IPA and MPA were similar to TA at clinically relevant low equimolar concentrations.
Elution of Metronidazole and Gentamicin from Polymethylmethacrylate Beads
Ramos, Jose Rafaelix (Virginia Tech, 2003-05-02)
Ten polymethylmethacrylate (PMMA) beads containing metronidazole (3 concentrations); gentamicin sulfate; or metronidazole and gentamicin sulfate were immersed in 5 ml of phosphate buffered saline in triplicate. Eluent was replaced at specified time intervals for 1 day (1, 3, 6, 12 and 24 hours), daily, or weekly for 21 days. Antibiotic concentrations were measured by high performance liquid chromatography. Changes in antibiotic bioactivity attributable to polymerization or co-polymerization of the antibiotics with PMMA, ethylene oxide sterilization, and storage of antibiotic-impregnated PMMA (AIPMMA) beads containing metronidazole were evaluated. Antibiotic elution patterns were similar for all groups. Day-1 elution for groups containing either metronidazole (3 concentrations) or gentamicin represented a mean 63% to 66% and 79% respectively of the 21-day total elution. Approximately 50% of the day-1 elution occurred during the first hour. The elution of metronidazole was dose-dependent. There was no significant difference in the total amount of antibiotic eluted from groups that had the saline changed daily versus weekly. The elution of metronidazole (day 3-21) and gentamicin (all days) was significantly greater when metronidazole and gentamicin were combined (p<0.05). Polymerization of PMMA was delayed in groups containing metronidazole. Neither polymerization nor co-polymerization of metronidazole and gentamicin with PMMA, gas-sterilization, or 2-month storage of beads containing metronidazole significantly affected antimicrobial bioactivity. Metronidazole elutes from PMMA. The frequency at which the saline was changed did not affect the rate of antibiotic elution. Co-polymerization of metronidazole and gentamicin sulfate in PMMA resulted in increased rates of elution. Intra-operative preparation of metronidazole-impregnated PMMA beads is not practical. However, prefabrication of metronidazole or metronidazole-gentamicin beads, gas-sterilization and storage for up to 2 months should not affect the efficacy of either antibiotic. The local delivery of biologically active metronidazole and gentamicin by elution from PMMA is feasible.
Endotoxin-induced microRNA expression in equine peripheral blood mononuclear cells
Parkinson, Nicholas J. (Virginia Tech, 2016-07-22)
The innate immune response to lipopolysaccharide (LPS) mediated by toll-like receptor 4 (TLR4) contributes substantially to the morbidity of equine gastrointestinal disease, neonatal sepsis and other diseases. MicroRNAs (miRNAs), small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in TLR4 signaling regulation in other species. The central hypothesis of this study was that LPS induces differential expression of miRNAs in equine peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy adult horses and cultured with LPS or medium only for 2, 4 and 8 hours. Concentrations of inflammatory cytokines were measured in supernatants by immunoassay. Illumina Next-Generation Sequencing of the miRNA transcriptome was performed in PBMCs at 0, 2 and 4 hours. Selected expression changes were verified by qRT-PCR. 327 mature miRNAs were detected in equine PBMCs. Only miR-155 was significantly upregulated by LPS. 9 miRNAs showed statistically significant expression changes with time. Tumor necrosis factor-α concentration was significantly higher in supernatants from LPS-treated cells than controls from 2 hours, while interleukin-10 and interferon-γ were increased at 8 hours. miR-155 expression was correlated to all three cytokines. These data provide a foundation for future research into miRNA involvement in equine inflammatory responses. miR-155 is the principal LPS-induced miRNA in horses. Bioinformatic target predictions support roles in regulation of innate and adaptive immune responses including TLR4 signaling, as in humans. It is thus likely to influence the acute inflammatory response to LPS. Further research will be necessary to establish its role in naturally occurring disease.
Evaluation of extracorporeal shockwave for treatment of horses with thoracolumbar pain
Burns, Lauren Trager (Virginia Tech, 2019-09-24)
The objective of this study was to evaluate effects of extracorporeal shockwave therapy (ESWT) on spinal mechanical nociceptive threshold (MNT) and multifidus muscle cross-sectional area (CSA) in horses with thoracolumbar pain. We hypothesized that ESWT would increase MNT and multifidus CSA. Twelve horses with thoracolumbar pain were included. Prior to treatment, each thoracolumbar spine was radiographed to document existing pathology. Horses received 3 ESWT treatments, 2 weeks apart (days 0, 14, 28). Palpation scores were documented (days 0, 45, 65) and ultrasonographic CSA of left and right multifidus was recorded at T12, T14, T16, T18, L3 and L5 (days 0, 45, 65). MNT was measured at T12, T14, T16, T18, L3 and L5 every 7 days (day 0-56). Change in MNT in 10/12 horses was significant at each timepoint compared to day 0 (P<0.05). MNT increased at all timepoints at 6 sites in 2/12, at 5 sites in 3/12, at 4 sites in 4/12 and at 1 site in 1/12 (P<0.05). MNT average percent increase from day 0-56 was 64% for T12-T18 and 29% for L3-L5. There was no statistical difference in MNT from day 35-56 (P=0.25). A bimodal analgesic trend was observed following ESWT. Degree of radiographic change was not associated with response to treatment and no significant change in multifidus CSA was observed. In conclusion, 3 treatments of ESWT 2 weeks apart raised MNT over a 56-day period in horses with back pain, but did not influence change in CSA of the multifidus.
Evaluation of the Harmonic Scalpel for Laparoscopic Bilateral Ovariectomy in Standing Horses
Duesterdieck, Katja Friederike (Virginia Tech, 2003-04-29)
Objective - To evaluate a surgical technique for performing laparoscopic bilateral ovariectomy in standing horses. Study Design - Experimental study. Animals or Sample population - 8 mares, age 2-20 years, weight 410-540 kg. Methods - Standing laparoscopic bilateral ovariectomy was performed in 8 mares with normal anatomy of the reproductive tract. The Harmonic Scalpel (an ultrasonically activated instrument) was used to transect the ovarian pedicle and to obtain hemostasis simultaneously. Necropsy was performed on 4 mares 3 days after surgery and 30 days following surgery on the remaining 4 mares. Gross and histopathologic evaluation of the ovarian pedicles was performed to characterize the effects of the Harmonic Scalpel on the transected tissue. Results - The Harmonic Scalpel achieved complete hemostasis of the vasculature of the ovarian pedicles in all mares. Median transection time for the ovarian pedicle was 28 minutes. Postoperative complications included transient fever in one mare, moderate subcutaneous emphysema in another, and incisional seroma formation in a third mare. Post-mortem examination 3 and 30 days postoperatively revealed no signs of generalized peritonitis, postoperative hemorrhage or adhesion formation. Mild to moderate acute inflammation, and scar formation with moderate chronic inflammation at the ovarian pedicle was found 3 and 30 days after surgery, respectively. Median depth of coagulation necrosis 3 days postoperatively was 2.87 mm. Conclusions - The Harmonic Scalpel appears to provide reliable hemostasis of the ovarian pedicle during elective laparoscopic ovariectomy in horses. Clinical Relevance - The Harmonic Scalpel represents a safe alternative to other means of hemostasis during elective laparoscopic ovariectomy in horses.
Experimental Evaluation of Urinary Bladder Marsupialization in Male Goats
May, Kimberly Anne (Virginia Tech, 1999-07-13)
Urinary bladder marsupialization has been successful in producing acceptable long-term resolution of clinical cases of obstructive urolithiasis in male goats. The purpose of this study was to evaluate the six-month outcome of urinary bladder marsupialization in male goats. The urinary bladders of six male goats free from systemic disease were marsupialized following induced urethral obstruction. Renal ultrasonography, complete blood count, and blood chemistry analysis were evaluated preoperatively (day 0), at 7 postoperative days, and at 30-day intervals until 180 postoperative days. Stomal diameter was recorded at each interval. Necropsy examination was performed on day 180 or when stomal stricture or death occurred. Stomal stricture occurred in one goat at 120 days, and another goat was found dead at 150 days. Necropsy of this goat revealed severe, suppurative cystitis. All goats developed mild urine scald dermatitis. All blood chemistry values remained within normal limits. Significant decreases in white blood cell count, serum creatinine, and stomal diameter were observed from day 0 to day 180. Except for the goat that died at 150 days, all urinary bladders were tubular in shape and the mucosa and serosa of all urinary tract organs appeared grossly normal at necropsy examination. Histologic evidence of chronic suppurative cystitis and chronic, mild, lymphoplasmacytic pyelitis was present in all goats. Culture of renal tissue yielded bacterial growth in three of six goats, and culture of a swab of the urinary bladder mucosa yielded bacterial growth in all animals. Although clinical signs of ascending urinary tract infection were not observed in goats with patent stomata, urinary bladder marsupialization may result in ascending inflammation or infection. Based upon the results of this study, urinary bladder marsupialization should be recommended with caution as the primary procedure in clinical cases.
Hand-assisted laparoscopic ovariohysterectomy in the mare
Delling, Uta (Virginia Tech, 2005-05-09)
Conventionally performed equine ovariohysterectomy (OHE) is a technically demanding surgery associated with a high degree of invasiveness and morbidity. Hand-assisted laparoscopic surgical technique allows introduction of a hand through a portal into the laparoscopic field to facilitate surgical manipulation while maintaining abdominal insufflation and laparoscopic visualization. The purpose of this study was to develop and evaluate a hand-assisted laparoscopic OHE technique for dorsally recumbent horses. The surgical technique was developed in terminal (2 mares) and subsequently evaluated in 6 survival procedures. Mares were fasted 48 hours, anesthetized and positioned in dorsal recumbency for laparoscopic surgery. A hand access device (Omniport) was placed in a caudal midline laparotomy followed by 4 laparoscopic portals. Transection of the ovarian pedicle and broad ligament was achieved using a combination of a laparoscopic stapling instrument, an ultrasonically activated instrument and endoscopic clips. The genital tract was exteriorized through the laparotomy, and the body of the uterus transected and sutured in conventional pattern. Horses were evaluated through postoperative day 14 when a post mortem evaluation was performed. Four mares recuperated well after surgery, 1 mare was euthanized due to bilateral femur fractures sustained during anesthetic recovery and another developed severe pleuropneumonia. At necropsy all but one abdominal incision was healing routinely. One mare had abscessed along the laparotomy incision and developed visceral adhesions. Uncomplicated healing of transected mesovarial, mesometrial and uterine remnants was observed in all recovered mares. Hand-assisted laparoscopic OHE technique represents a minimally invasive and technically feasible alternative for conventional OHE. Careful patient selection and preparation may reduce the complications observed in this study.
Health Care for Horses
Pleasant, R. Scott; Currin, Nancy (Virginia Cooperative Extension, 2009)
Provides a broad overview of health care for horses.
Investigation into the use of an epidural morphine sulfate and detomidine hydrochloride combination in horses: Part 1: efficacy in alleviation of hindlimb pain, Part 2: long-term systemic and local effects
Sysel, Annette M. (Virginia Tech, 1996)
In Part 1, amphotericin B-induced synovitis of the left tarsocrural joint was used to create hindlimb lameness in 11 horses. Caudal epidural catheters were placed and advanced to the lumbosacral region. Baseline heart and respiratory rates were recorded and horses were videotaped at a walk and trot. Treated horses received 0.2 mg/kg morphine sulfate and 30 ug/kg detomidine hydrochloride through the epidural catheter; control horses received an equivalent volume of physiologic saline solution through the catheter. At hourly intervals after epidural injection for a total of 6 hours, heart and respiratory rates were recorded and horses were videotaped walking and trotting. At the end of the observation period, video recordings were scrambled onto a master videotape. Lamenesses were scored by 3 investigators. Lameness grades, heart rates and respiratory rates were compared. There was a significant decrease in lameness grades after treatment with epidural morphine and detomidine. Initially, heart rates significantly increased in control horses and decreased in treated horses. A similar trend occurred for respiratory rates. In Part 2, caudal epidural catheters were used to administer injections to 10 horses every 12 hours for 14 days. Treated horses received 0.2 nlg/kg morphine sulfate and 30 ug/kg detomidine hydrochloride, and control horses received an equivalent volume of physiologic saline solution. Body weights were recorded on days 1 and 14. Rectal temperature, heart rate, respiratory rate and gastrointestinal motility were recorded twice daily, and daily hay and water consumption was measured. Horses were euthanatized day 15. Atlanto-occipital cerebrospinal fluid samples were submitted for bacteriologic culture and determination of white and red blood cell counts and protein and glucose concentrations. Post mortem examinations were performed and representative samples of the spinal cord and surrounding tissues were taken from cervicothoracic, thoracolumbar, lumbosacral, sacral and catheter entry point regions. Spinal tissue segments from these regions were graded for histologic degree of inflammation and fibrosis. Cerebrospinal fluid values and spinal tissue segment inflammation and fibrosis grades were compared between control and treated horses, and between all 10 catheterized study horses and 6 uncatheterized horses. No problems were encountered with epidural catheter maintenance or injection. No significant difference was identified in body weight change, daily variables or hay and water consumption between control and treated horses. All cerebrospinal fluid cultures were negative for growth. No significant difference in cerebrospinal fluid values or spinal tissue inflammation or fibrosis grades for any segment was demonstrated between control and treated horses. However, when compared to uncatheterized horses, cerebrospinal fluid red blood cell counts were marginally higher and protein concentrations were significantly higher in catheterized horses. As well, lumbosacral and sacral spinal tissue segment inflammation grades and sacral segment fibrosis grades were significantly higher in catheterized compared to uncatheterized horses. Results of these studies indicate that an epidural combination of morphine and detomidine provides profound hindlimb analgesia in horses and is not associated with apparent adverse systemic effects. Localized epidural inflammation and fibrosis appear to be catheter-related.
Occlusion of the Internal Carotid Artery of Horses: Evaluation of a Technique Designed to Prevent Epistaxis Caused by Guttural Pouch Mycosis
Cheramie, Hoyt Stephen (Virginia Tech, 1998-06-19)
In six, healthy, adult horses, the origin of the left internal carotid artery was isolated via a modified hyovertebrotomy approach. Normograde blood flow was occluded by placement of a tourniquet on the artery near its origin. Lumenal access was gained through placement of a distally directed introducer sheath and retrograde blood flow from the cerebral arterial circle was confirmed. An 8.5 mm diameter detachable latex balloon loaded onto a carrier catheter and placed within a guiding catheter was introduced into the internal carotid artery through the introducer sheath and advanced to the target occlusion site (the proximal curve of the sigmoid flexure of the internal carotid artery). The balloon was inflated with 0.5 ml of a radiopaque solution. Correct placement and inflation of the balloon were confirmed by intraoperative radiography. The balloon was then released and the guiding and carrier catheters withdrawn. Immediate embolization of the distal internal carotid artery was determined by lack of retrograde blood flow through the introducer sheath. The introducer sheath was withdrawn from the vessel and the proximal tourniquet was replaced with two ligatures. Horses were euthanized on day 30 and detailed gross and histopathologic examinations were performed. The balloons were easily placed into the target site and produced immediate occlusion of retrograde flow from the cerebral arterial circle. All balloons remained inflated in their original position throughout the study period. Mature thrombus formation and absence of clinically significant inflammation were consistent findings in all occluded internal carotid arteries at gross necropsy and histologic examination.
A pharmacokinetic and pharmacodynamic study of pioglitazone in a model of induced insulin resistance in normal horses
Wearn, Jamie Macquarie (Virginia Tech, 2010-05-28)
Equine Metabolic Syndrome (EMS) is a unique condition of horses characterized by adiposity, insulin resistance, and an increased risk of laminitis. Reducing insulin resistance may decrease the incidence of laminitis in horses with EMS. Pioglitazone, a thiazolidinedione class of anti-diabetic drug, has proven efficacy in humans with type 2 diabetes, a syndrome of insulin resistance sharing some similarities with EMS. The ability of pioglitazone to influence insulin sensitivity in an endotoxin-infusion model of induced insulin resistance was investigated. Our hypothesis was that piogltiazone would preserve insulin sensitivity in a model of induced insulin resistance. The specific aims were to investigate the pharmacokinetics and pharmacodynamics of pioglitazone in an endotoxin infusion model of insulin resistance. 16 normal adult horses were enrolled. Pioglitazone was administered to 8 horses (1 mg/kg, PO, q24h) for 14 days, and 8 horses served as their controls. Liquid chromatography with tandem mass spectroscopy was used to quantitate plasma concentration of pioglitazone. A frequently sampled intravenous glucose tolerance test with minimum model analysis was used to compare indices of glucose and insulin dynamics prior to, and following, endotoxin infusion in horses treated with pioglitazone and their controls. Parameters of clinical examination and lipid metabolism were compared prior to, and following, endotoxin administration. Pioglitazone administered orally at 1 mg/kg q 24 h resulted in plasma concentrations lower, and more variable, compared to those considered therapeutic in humans. No significant effect of drug treatment was detected on clinical parameters or indices of insulin dynamics or lipid homeostasis following endotoxin challenge.
Porcine urinary bladder matrix in an in vitro equine model of tenogenesis
Khatibzadeh, Sarah M. (Virginia Tech, 2019-08-22)
Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone.
Recombinant Equine Interleukin-1 Induced Models of Equine Joint Disease
Takafuji, Vivian Ann (Virginia Tech, 2003-11-13)
Osteoarthritis (OA) is a debilitating disease of joints that afflicts horses of all ages and breeds and can result in lameness, suboptimal performance, and decreased quality of life. The pro-inflammatory cytokine interleukin-1 (IL-1) has been associated with the initiation and pathogenesis of joint disease. In part, this occurs by induction of proteases and oxidative pathways that contribute to the degradation of structural components of the articular cartilage extracellular matrix. Elucidating the complex macromolecular and molecular effects of IL-1 on articular tissues may further our understanding of the roles of IL-1 and inflammation in OA pathobiology. Full-length gene sequences encoding three recombinant equine interleukin-1 proteins (EqIL-1a, EqIL-1b, and EqIL-1 receptor antagonist), were previously cloned and expressed in-vitro. The objectives of this dissertation were to 1) establish EqIL-1 induced experimental models of equine OA, and 2) to investigate specific IL-1-induced immuno-inflammatory responses. Effects of EqIL-1 on articular cartilage explant proteoglycan metabolism and synthesis of a downstream inflammatory product, prostaglandin E2, established culturing conditions and furthered the rationale to use EqIL-1 in the in-vitro modeling of early joint disease. A customized cDNA array was used to profile changes in mRNA levels resulting from EqIL-1 treatments of cultured articular cartilage chondrocytes. EqIL-1a induced elevated mRNA levels corresponding to six genes after 1 hour relative to media control chondrocytes (p<0.05). EqIL-1b increased transcript levels of seven genes after 6 hours (p<0.0004); 102 additional transcripts were elevated > 2-fold over controls. A subset of the array-generated data was verified using optimized reverse transcriptase-PCR amplification. Results of principal component analysis indicate co-regulation of EqIL-1 induced transcript levels to relate to chondrocyte differentiation and cell-cycle processes. Subtractive hybridization-PCR identified 148 differentially expressed cDNAs in synovium resulting from a 6-hour intra-articular EqIL-1b injection. Combined results demonstrate the potent bioactivity of our equine IL-1 proteins and support the argument for crucial roles of IL-1 in pro-inflammatory processes and cytokine imbalances underlying early OA pathogenesis. These results add to the current knowledge of IL-1 modulated transcription that may precede ECM catabolic processes characteristic of OA. The culture systems, assays, and techniques for gene expression analysis may be useful for future studies attempting to elucidate macromolecular and transcriptional events underlying inflammatory-associated joint disease processes in horses. Reported information may further efforts toward improved diagnostic and preventive strategies and development of anti-IL-1 directed therapies.
The Use of Steered Ileo-cecal Valve Cannulated Pigs to Evaluate the Effects of Adding Phytase or Beta-mannanase to the Diet on Amino Acid, Mineral and Energy Utilization
Radcliffe, John Scott (Virginia Tech, 2001-04-13)
Forty-six barrows fitted with steered ileo-cecal valve cannulas were used in four experiments to evaluate the effects of supplementing swine diets with microbial phytase or beta-mannanase on the apparent ileal (AID) and/or apparent total tract digestibility (ATTD) of amino acids, N, Ca, P, DM and energy. In Exp. 1, the addition of phytase to low CP corn-soybean meal based diets increased the AID of Ca (P < .01), P (P < .001), and all amino acids (P < .10) measured except Leu, Ser, Pro, Met, His and Tyr. In Exp. 2, the addition of microbial phytase to corn-soybean meal, corn-soybean meal-wheat middlings, or corn-soybean meal-meat and bone meal based diets resulted in increased AID of Ca and P, but had no effect (P > .1) on amino acid digestibilities. Diet type affected all digestibility measurements, but did not affect the efficacy of supplemental phytase. In Exp. 3, the addition of microbial phytase to corn-wheat-soybean meal, corn-wheat-cannola, or sorghum-corn-soybean meal based diets led to an increased ( P <.05) AID of P, Asp, Thr, Ser, Ala, Tyr, Phe, Lys and Arg. In Exp. 4, the addition of beta-mannanase to corn-soybean meal based swine diets led to an increased AID of DM and ATTD of energy. In addition, the AID of all amino acids measured were increased numerically, with many of these values approaching significance. The results of these studies demonstrate that supplementing pig diets with phytase or beta-mannanase, results in an increased digestibility of certain dietary components due to the breakdown of anti-nutritive compounds in the diet.

Browsing by Author "Pleasant, R. Scott"

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