VTechWorks Repository :: Browsing by Author "White, Nathaniel A. II"

Browsing by Author "White, Nathaniel A. II"

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Apoptosis in the equine small intestine following experimental ischemia-reperfusion injury
Nagy, Amy Dae (Virginia Tech, 2008-05-23)
This study was aimed at characterizing the apoptotic response equine small intestine subjected to experimental ischemia-reperfusion injury and determining if use of an angiotensin converting enzyme inhibitor (enalaprilat) would ameliorate the apoptotic response. It was designed to determine if mucosal epithelial cells undergo apoptosis during the ischemia phase and also examined if apoptosis is significantly exacerbated by reperfusion. It also investigated whether administration of enalaprilat decreased reperfusion injury secondary to reduced enterocyte apoptosis. Injury was induced using a low flow model of I-R. During celiotomy a single loop of jejunum was isolated and arterial flow decreased to 20% of baseline for one hour and complete occlusion for a second hour. Reperfusion was monitored for 3 hours. A control group (n=6) were not treated while the treatment group (n=6) received 0.5 mg/kg enalaprilat in 0.9% NaCl immediately following ischemia. Jejunal samples were taken prior to the induction of ischemia, immediately post-ischemia and at 1, 2 and 3 hours of reperfusion. Samples were evaluated for gross tissue pathology with standard hematoxylin and eosin staining, the presence of apoptotic cells via TUNEL staining, and gene expression of three apoptosis related genes (bax, bcl-2, p53) using qPCR. Serum enalaprilat and ACE concentrations were determined from blood samples drawn concurrent with jejunal sampling using HPLC/MS and standard HPLC. Plasma enalaprilat concentrations were comparable to previous reports in awake horses. Enalaprilat appeared to have no effect on serum ACE concentrations, however a significant spike in ACE concentration occurred in the treatment group at 1 hour of reperfusion (P=0.0001). Grade of mucosal damage was not significantly different between control and treatment groups at any time point. Subjectively apoptotic index appeared to be higher in the treatment group at end ischemia and during reperfusion. There were no changes in expression of p53 or bcl-2 in either group. Bax expression was significantly decreased (P= 0.02) in the control group at 2 hours of reperfusion. Based on our data administration of an ACE inhibitor during anesthesia in horses with an ischemic segment of intestine confers no protective benefit and may be associated with increased intestinal injury and apoptosis. Lack of expression of p53, bax and bcl-2 suggests another apoptotic mechanism in equine ischemic intestine.
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.
Biomechanical analysis of a novel suture pattern for repair of equine tendon lacerations
Everett, Eric K. (Virginia Tech, 2011-04-14)
Flexor tendon lacerations in horses are traumatic injuries that can be career ending and life threatening. In the horse, a tendon repair must withstand the strains placed on the tenorrhaphy by immediate weight bearing and locomotion post-operatively. Despite the use of external coaptation, such strains can lead to significant gap formation, construct failure, longer healing time and poor quality of the healed tendon. Similar to equine surgery, gap formation and construct failure are common concerns in human medicine, with early return to post-operative physiotherapy challenging the primary repair. Early return to exercise and decreased gap formation has been shown to reduce adhesion formation. Based on these concerns, the ideal tenorrhaphy suture pattern for equines would provide: 1) high ultimate failure load, 2) resistance to gap formation, 3) minimal alteration in blood supply, and 4) minimal adhesion formation. Historically, various suture patterns and materials have been evaluated for human and equine flexor tendon repair. Results of equine studies suggest the three-loop pulley pattern (3LP) compares favorably to other patterns and is recommended for primary tenorrhaphy. However, this pattern still experiences significant gap formation and can result in failure. As a result, a technique which decreases the problems inherent in the 3LP is warranted for tenorrhaphy of equine flexor tendons. A review of the human literature highlights certain characteristics of the tenorrhaphy that may improve results including core purchase length and suture loop characteristics. Optimization of these tenorrhaphy characteristics can increase tenorrhaphy performance and patient outcome. The six-strand Savage technique (SSS) is a pattern routinely used in human hand surgery for tendon repair, and possesses high ultimate failure load and resistance to gap formation that may be beneficial for application in equine tendon repair. This study compared a novel tenorrhaphy pattern for horses, the SSS, with the currently recommended pattern, the 3LP, in an in vitro model. We hypothesize the SSS will fail at a higher ultimate load, resist pull through, and resist gap formation better than the 3LP. All testing used cadaveric equine superficial digital flexor tendons from horses euthanized for reasons other than musculoskeletal injury. All testing was approved by the IACUC. The two techniques were applied to cadaveric equine superficial digital flexor tendons. The same investigator performed all repairs (EE). Biomechanical properties were determined in a blinded, randomized pair design. Ultimate failure load, mode of failure and load required to form a 3mm gap were recorded on an Instron Electropuls materials testing system. Gap formation was determined using synchronized high-speed video analysis. Results are reported as mean + standard deviation. Statistical comparisons were made using Student's T test, with significance set at p<0.05. The tenorrhaphies were tested for their ultimate failure load and failure mode. The mean failure load for the SSS construct (421.1 ± 47.6) was significantly greater than that for the 3LP repaired tendons (193.7 ± 43.0). Failure mode was suture breakage for the SSS constructs (13/13) and suture pull through for the 3LP constructs (13/13). The maximum load to create a 3mm gap in the SSS repair (102.0N ± 22.4) was not significantly different from the 3LP repair (109.9N ± 16.0). The results of the current study demonstrate that the SSS tenorrhaphy has a higher ultimate failure load and resistance to pull through than the 3LP. The biomechanical properties of the SSS technique show promise as a more desirable repair for equine flexor tendons. However, in vivo testing of the effects of the pattern on live tissue and in a cyclic loading environment is necessary before clinical application of the pattern is recommended.
Characterization of Apoptotic Cells in Equine Proximal Suspensory Desmitis
Hewes, Christina Andrea (Virginia Tech, 2006-05-30)
Suspensory desmitis is a common problem and affects a broad cross section of equine athletes in various disciplines. For this study, the proximal portion of the suspensory ligament was collected from 6 horses without suspensory ligament injury (16 ligaments) and 4 horses with degeneration of the suspensory ligament (11 ligaments). Specimens were collected immediately after euthanasia and placed in neutral-buffered 10% formalin. The tissue was fixed, sectioned, and stained with hematoxylin and eosin (H&E), Masson's trichrome, and for apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. Histological changes in the abnormal ligaments included mineralization, fibroplasias, neovascularization, collagen degeneration, and significant architecture disruption in 2 ligaments. There was a trend for increased apoptosis in the injured ligaments compared to the normal ligaments.
Comparison of bone marrow mesenchymal stem cells and tendon progenitor cells cultured on collagen surfaces
Brown, James Augustus (Virginia Tech, 2010-04-29)
Tendon injuries are a significant cause of morbidity in performance horses with superficial digital flexor tendon injury reported to represent up to 43% of overall Thoroughbred racehorse injuries. Natural repair is slow and results in inferior structural organization and biomechanical properties and, therefore, reinjury is common. The inability of tendon to regenerate after injury, or to heal with mechanical properties comparable to the original tissue, is likely attributable to low vascularity and cellularity of the tissue, low number of resident progenitor cells, and healing under weight-bearing conditions. Strategies to improve tendon healing have focused on enhancing the metabolic response of tenocytes, modulating the organization of the newly synthesized extracellular matrix, or administering progenitor cells to enhance repair. Significant research effort has been directed at the use of adult mesenchymal stem cells as a source of progenitor cells for equine tendon repair and recent clinical applications have utilized adult autologous stem cells derived either from adipose tissue or bone marrow aspirates. Isolation of a homogenous population of stem cells from bone marrow is time-consuming, and there is much variation in cell numbers, cell viability and growth rates among samples. Recently, a population of progenitor cells has been isolated from equine flexor tendons, thus providing an alternative source of progenitor cells from the target tissue for therapeutic intervention. The interaction between cells and the extracellular matrix (ECM) is an important factor in regulation of cell function. Proliferation, migration, differentiation and gene expression of many cell types are altered by adhesion to and interaction with matrix proteins and the extracellular environment. Tendon progenitor cells reside within a niche that comprises primarily parallel collagen fibers, and this niche plays an important role in regulating their function and differentiation. Culture conditions replicating this environment could be beneficial for both cell growth and matrix gene expression. The objectives of the study were to compare cell growth kinetics and biosynthetic capabilities of bone marrow mesenchymal stem cells (BMMSCs) and tendon derived progenitor cells (TPCs) cultured on commercially available bovine, highly purified bovine, porcine, and rattus collagen sources and standard tissue culture surfaces. We hypothesized that collagen type I matrix would preferentially support TPC proliferation and up regulate gene expression for collagens and organizational components of tendon and therefore provide a culture system and progenitor cell type with advantages over the current practice of BMMSC expansion on standard cell culture plastic surfaces. Cells were isolated from 6 young adult horses, expanded, and cultured on collagen-coated tissue culture plates, and no collagen control for 7 days. Samples were analyzed for cell number on days 4 and 7, and for mRNA expression of collagen type I, collagen type III, cartilage oligomeric matrix protein (COMP), and decorin on day 7. Glycosaminoglycan (GAG) synthesis was analyzed on day 7. Differences of cell number between collagen groups and cell type, and in gene expression and GAG synthesis between collagen groups and cell types, were evaluated by use of mixed-model repeated measures ANOVA. Pair-wise comparisons were made on significant differences identified with ANOVA using Tukey's post hoc test. Statistical significance was set at P<0.05. A statistical significant (P=0.05) increase in cell number for TPCs grown on rattus collagen versus control on day 4 was observed. No difference in GAG synthesis or expression of collagen type I, collagen type III, COMP or decorin mRNA was observed between collagen groups and non-collagen controls for either cell type on day 7. TPCs cultured on all collagen types yielded more cells than similarly cultured BMMSCs on day 4, but only porcine collagen was superior on day 7. TPCs synthesized more GAG than BMMSCs when cultured on control surfaces only. BMMSCs expressed more collagen type I mRNA when cultured on control, porcine and highly-purified collagen, and more collagen type III when cultured on control, porcine, highly-purified collagen, and rattus collagen, than TPCs. Tendon-progenitor cells expressed significantly more COMP when cultured on control and all collagen types, and decorin when cultured on porcine, highly purified bovine and bovine collagen when compared to BMMSCs. The results of this study revealed an advantage to culturing TPCs on randomly organized rattus collagen during the early growth phase. The beneficial effects of collagen-coated surfaces on cell proliferation is likely related to increased surface area for attachment and expansion provided by the random collagen matrix, and/or collagen-cell interactions. Tendon progenitor cells showed superior growth kinetics and expression of the matrix organizational components, COMP and decorin, than similarly cultured BMMSCs that expressed more collagen types III and I. TPCs synthesize more GAG compared to BMMSCs when cultured on plastic surfaces and there was no induction by collagen. Tendon progenitor cells should be considered as an alternative source of progenitor cells for injured equine tendons. Further in vitro studies characterizing factors that influence gene expression of both cell types is warranted.
Comparison of healing of full-thickness cartilage vs. full- thickness cartilage and subchondral bone defects in the equine third carpal bone
Hanie, Elizabeth Anne (Virginia Tech, 1991-05-05)
The quality of repair tissue produced by defects which exposed the subchondral bone· plate and those which penetrated the subchondral bone plate to expose the subchondral marrow spaces was compared in the equine third carpal bone. Specimens were examined four and six months after surgery. Exposure of the subchondral bone plate resulted in a deep layer of fibrocartilage and a superficial layer of fibrous connective tissue. Exposure of the subchondral marrow spaces resulted in a deep layer of hyaline-like cartilage, an intermediate layer of fibrocartilage and a superficial layer of fibrous connective tissue. Degenerative joint disease occurred in all joints. Results were similar between four- and six-month specimens. Further studies are needed before removal of the subchondral bone plate can be recommended for clinical treatment of full-thickness articular cartilage lesions.
Cross-Sectional Survey of Horse Owners to Assess Their Knowledge and Use of Biosecurity Practices for Equine Infectious Diseases in the United States
White, Nathaniel A. II; Pelzel-McCluskey, Angela (MDPI, 2023-11-17)
Horses are transported in the United States more than any other livestock species and co-mingle at various events; therefore, they are considered to be at an increased risk for infectious disease transmission. The fragmented movement of horses combined with numerous sites of co-mingling makes tracing the potential spread of a disease outbreak a necessary part of an infection control plan, both locally and nationally. The cross-movement of personnel with horses and the persistence of endemic diseases make biosecurity implementation an ongoing challenge. Although many of the risks for infection are known, there is limited documentation about the usefulness of prospective control measures. The objective of this survey was to determine horse owners’ understanding and knowledge of biosecurity practices for preventing infectious diseases in the United States. Questions covered owner demographic information, including horse use which was divided into 10 categories as follows: Pleasure/Trail Riding, Lessons/School, Western Show, English Show, Breeding, Farm/Ranch, Retired, Racing, Driving and Other. The survey was distributed by sending requests to a list of horse owner organizations, which then sent emails to their members. The email request described the survey and provided a website link to start the survey. A total of 2413 responses were collected. Analysis of the results included cross-tabulation to identify significant differences in biosecurity knowledge and awareness by horse use. Significant differences by horse use were identified for vaccination, biosecurity planning, use of isolation, disease risk, monitoring for diseases, co-mingling of horses, sanitation, medical decision making and health record requirements for horse events. In summary, the results suggest that most owners are not highly concerned about the risk of disease or the use of biosecurity. There are several biosecurity applications and techniques which can be increased and will benefit horse health and welfare. These include reliance on temperature monitoring, isolation of new horses at facilities, risks of horse mingling, entry requirements such as vaccination and health certificates at events, and an emphasis on having biosecurity plans for facilities and events where horses co-mingle. The information from this study will be used to create tools and information that horse owners and veterinarians can use to implement appropriate biosecurity practices for different types of horse uses and events.
Detection of apoptotic cells in horses with and without gastrointestinal disease
Rowe, Emma L. (Virginia Tech, 2003-04-25)
A study was performed to identify apoptotic cells in the equine intestine and to determine if the occurrence of apoptosis is affected by gastrointestinal disease and tissue layer of intestine. Samples of intestine were collected from 38 horses that underwent surgery or were humanely destroyed for small or large bowel obstruction, strangulation or distension. Samples were also taken from 9 horses which were humanely euthanized for reasons other than gastrointestinal disease or systemic disease. Specimens were collected at surgery from intestine involved in the primary lesion, distant to the primary lesion, or at necropsy from several sites including the primary lesion. Tissues were fixed, serially sectioned and stained with hematoxylin and eosin (H&E) and for apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. The number of apoptotic cells per high power field were counted in the mucosa, circular muscle, longitudinal muscle and serosa for each sample of intestine. Apoptotic staining nuclei were seen in all layers of intestine. An increased number of apoptotic cells were found in the circular muscle of the intestine from horses with simple obstruction. Intestine distant from the primary strangulating lesion had higher numbers of apoptotic cells than intestine distant from a simple obstruction lesion or intestine taken at the site of a strangulating or simple obstructive lesion. Intestine from horses with obstructing or strangulating lesions in the small intestine and large colon has increased numbers of apoptotic cells. Further investigation is required to determine whether increased apoptosis affects intestinal function.
Determination of cardiac output across a range of values in horses by M-mode echocardiography and thermodilution
Moore, Donna Preston (Virginia Tech, 2004-02-12)
Determinations of cardiac output (CO) by M-mode echocardio-graphy were compared with simultaneous determinations by thermodilution in 2 conscious and 5 anesthetized horses. A range of cardiac outputs was induced by use of a pharmacological protocol (dopamine, 4 ug/kg/min, dobutamine, 4 ug/kg/min, and 10 ug/kg detomidine plus 20 ug/kg butorphanol, in sequence). Changes from baseline CO in response to each drug were evaluated, and data was analyzed to determine whether there were any interactions between drug treatment and measurement method. The mathematical relationship between CO as determined by M-mode echocardio-graphy (COecho) and as determined by thermodilution (COTD) was described and used to predict COTD from COecho. The 2 methods were compared with respect to bias and variability in order to determine the suitability of COecho as a substitute for COTD . Sources of the variability for each method were determined. Determination of CO by either method in standing horses was prohibitively difficult due to patient movement. The pharmacologi-cal protocol was satisfactory for inducing a range of cardiac outputs for the purpose of method comparison; however, use of dopamine did not offer any additional benefit over the use of dobutamine and was generally less reliable for increasing CO. Inclusion of detomidine provided an additional change in CO but did not increase the overall range of CO over that produced by halothane and dobutamine. COecho and COTD were significantly related by the predictive equation COTD = (0.63 +/- 0.157) x COecho + (16.6 +/- 3.22). The relatively large standard errors associated with COecho measurements resulted in a broad 95% prediction interval such that COecho would have to change by more than 100% in order to be 95% confident that the determined value represents true hemodynamic change. COecho underestimated COTD by a mean of 10 +/- 6.3 l/min/450 kg. The large standard deviation of the bias resulted in broad limits of agreement (-22.3 to +2.3 l/min/450 kg). Measurement-to-measurement variability accounted for 28% of the total variation in COTD values and 64% of the total variation in COecho values. Results might be improved if the mean of 3-5 consecutive beats was used for each measurement, but as determined in this experiment, COecho is too variable to have confidence in its use for precise determinations of CO.
Evaluation of the microcirculation of the equine small intestine following intramural distention and reperfusion
Dabareiner, Robin Marie (Virginia Tech, 1992-04-05)
The effects of intraluminal distention (25 cm H₂O, 120 minutes) and subsequent decompression (60 minutes) on the intramural vascular patterns of the equine small intestine was evaluated in 7 anesthetized horses. The vascular system of experimental and control segments were injected with a blue-colored radiopaque medium for microangiography and histology or a diluted methyl methacrylate (MERCOX CL-2B) for scanning electron microscopy. The distended segments had shortened villi that were separated by expanded crypts and mesothelial cell loss, neutrophil infiltration and edema in the seromuscular layer. The number of filled vessels was decreased in the seromuscular layer and to a lesser extent in the mucosal layer in the distended segments compared to controls. Following reperfusion, the morphologic lesions progressed and the number of observed vessels increased in all layers; however the vascular density did not return to the pre distention state. This study identifies altered intramural vascular patterns in the equine jejunum during luminal distention and reperfusion.
Evaluation of the toxic effects of eltenac (4-((2,6-dichlorophenyl) amino)-3-thiophene acetic acid), a nonsteriodal anti-inflammatory drug, in horses
Goodrich, Laurie Ruth (Virginia Tech, 1996-05-15)
A controlled study was performed to determine the potential toxic effects of the new nonsteroidal anti-inflammatory drug, eltenac (4-[(2,6-dichlorophenyl) amino]-3-thiopheneacetic acid), in horses. Four treatment groups were formed, each composed of 6 horses. The drug was injected intravenously, once daily, at a dose rate of 0.5 mg/kg, 1.5mg/kg or 2.5 mg/kg for 15 days. A control group was injected with sterile saline solution. Parameters assessed were hay and water consumption, daily clinical observations (evaluation of attitude, mentation, pulse and respiratory rate, fecal consistency, skin condition, and color and hydration of mucous membranes), physical examinations, complete hemogram, coagulation profiles, serum biochemical profiles, urinalysis, gastroscopic examinations and gross post-mortem and histopathological examinations on all organ systems. All examiners were blinded to group assignment and dosage levels until the completion of the study. A few glandular gastric ulcers, mild in severity, developed in seven animals during the treatment period. This occurred more often in horses treated with high doses of eltenac (P=.02). A dose dependent change of WBC count and neutrophil count were noted. Total protein, albumin and globulin levels had dose dependent decreases. One horse in the high dose group (2.5mg/kg) developed ventral edema as well as hypoproteinemia. N one of the horses in any of the dosage groups exhibited depression or anorexia. Gross post-mortem and histologic examination did not reveal any signs of drug related gastrointestinal, renal or hepatic abnormalities. Minimal toxic effects of eltenac given intravenously were greatest in horses treated with 2.5 mg/kg of the compound for 15 days.
Evaluation of Two Techniques of Cancellous Bone Grafting of Experimental Subchondral Bone Cysts in the Medial Femoral Condyles of Horses
Farnsworth, Kelly Dee Jr. (Virginia Tech, 1998-06-10)
Cylindric (10 mm diameter X 15 mm deep) osteochondral defects were created in the medial femoral condyles of 5 horses to mimic clinical cases of subchondral bone cysts after debridement. The defects were created with either a standard square ended drill bit or a compaction drill bit. The compaction drill bit compresses bone laterally and distally creating a dense wall and floor. Twelve-mm sternal cancellous cylinders were compressed to 9.25 mm and inserted into the femoral defects where they were presumed to expand and tighten the fit. The end result was sternal cancellous bone that exactly fit the femoral defects. Fluorochrome bone labels were used to confirm the origin of bone present in the defects at necropsy, which was performed after 6 months. Successful graft incorporation occurred in 3 of the compacted and 2 of the noncompacted defects. The surfaces of the successful cancellous bone grafts contained predominately fibrocartilage. The unsuccessful noncompacted defects expanded laterally and deeply into the parent bone epiphysis while the unsuccessful compacted defects remained confined to the originally created size.
A farm-based prospective study for equine colic risk factors and risk associated events
Tinker, Mary Kay (Virginia Tech, 1995)
Improved definition of risk factors for equine colic is necessary to develop effective interventions to reduce colic incidence. A one-year prospective study was conducted to estimate colic incidence and to identify risk factors. Farms with greater than 20 horses were randomly selected from two adjacent counties of Virginia and Maryland. Management information was recorded by questionnaire for 31 farms with 1427 horses. Owners kept calendars to record occurrence of specified events. Colic was reported by the owner when a horse exhibited signs of abdominal pain. The incidence of colic was 10.6 colic cases per 100 horse-years, based on 104 cases per 983.5 horse-years. Twenty-five deaths occurred from all causes, the proportional mortality rate of colic was 7/25 (28%). Risk factors were analyzed by logistic regression at the farm-level and the horse-level with farm as a random effects variable. No farm-level variables were significant. Significant horse variables were: age 2-10 years, odds ratio (OR)=2.8 (95% confidence interval, 1.2-6.5); previous colic, OR=3.6(1.9-6.8); changes in concentrate feeding during the year, OR=3.6(1.6-5.4); more than one change in hay feeding during the year, OR=2.1(1.2-3.8); feeding high levels of concentrate (>2.5 kg/day dry matter, OR=4.8(1.4-16), >5 kg/day dry matter, OR=6.3(1.8-22)); and vaccination with monocytic ehrlichiosis vaccine during the study, OR=2.0(1.8-22). Feeding whole grain with or without other concentrates had less risk than diets without whole grain included. Variables related to concentrate feeding frequency or concentrate type could be substituted for the concentrate level variable. A nested analysis examined risk for the time period following an event. The odds ratio was determined for the proportion of cases with an event within 14 days prior to the colic-date, relative to the proportion of horses without colic with an event within 14 days of a date chosen at random from the observation time. Weather events were analyzed for the three days before the colic or assigned date. Foaling was analyzed for three time periods: before, 0-60 and 60-150 days post-foaling. Significant events were recent vaccination, OR=3.31(1.9-6.0); recent transport, OR=3.3(1.2-5.5); 60-150 days post-foaling, OR=5.9(1.8-13); and recent fever, OR=20(2.5-169). Snow on the day of the colic, OR=2.8(1.0-7) and humidity <50% the day before the colic OR=1.6(1.0-2.9) were marginally significant.
Hydration of Colonic Ingesta and Feces in Horses Fed Large Grain Meals or Treated with Enteral Fluid Therapy, Saline Cathartics and Intravenous Fluid Therapy
Lopes, Marco A. F. (Virginia Tech, 2002-09-30)
Systemic hydration, plasma electrolytes, ingesta and fecal hydration and gastrointestinal passage of cobalt (after CoEDTA administration via nasogastric tube) in horses fed large grain meals or treated with enteral fluid therapy, IV fluid therapy and enteral laxatives were investigated. In the first study, 0.9% NaCl (10 L/h/8h) was administered slowly via a small-bore nasogastric tube or as 10 L boluses via a large-bore nasogastric tube to four normal horses. In the other studies, horses with a right dorsal colon fistula were used. To create the right dorsal colon fistula, a cannula with 5 cm internal diameter was implanted 2 to 6 weeks after a right dorsal colopexy had been created. Six horses with the right dorsal colostomy were alternately used to test three feeding regimes for 48 h: 1- hay free choice; 2- hay free choice plus 4.5 kg of sweet feed twice daily after a period of 5 days of adaptation; 3- sudden change from hay to hay plus sweet feed. Seven horses with the right dorsal colostomy were alternately used to test 6 experimental conditions while fasted for 24 h: 1- control (no treatment), 2- enteral MgSO4 (1 g/kg), 3- enteral Na2SO4 (1 g/kg), 4- IV lactated Ringer's solution (5 L/h/12 h), 5- enteral water (5 L/h/12 h), 6- enteral electrolyte solution (5 L/h/12 h). In the last study, four horses with the right dorsal colostomy were alternately treated with enteral electrolyte solution (10 L/h/6h) and enteral MgSO4 (1 g/kg) plus IV fluid therapy (10 L/h/6h). Despite the administration regimen, enteral administration of 0.9% NaCl produced diarrhea, hypernatremia and hyperchloremia. Colostomy allowed serial collection of large ingesta samples. Grain ingestion did not change PCV or plasma protein, but affected plasma electrolytes and produced dehydration of ingesta and formation of frothy ingesta. Fasting delayed gastrointestinal transit. Enteral fluid therapy was the most effective treatment in promoting ingesta hydration. Enteral water, MgSO4, Na2SO4, IV fluid therapy and enteral MgSO4 plus IV fluid therapy were either ineffective in promoting ingesta hydration or produced marked plasma electrolyte imbalance. These findings support the use of enteral fluid therapy in horses with gastrointestinal impaction.
Investigation into the Presence of Helicobacter in the Equine Stomach by Urease Testing and Polymerase Chain Reaction and Further Investigation into the Application of the 13C-Urea Blood Test to the Horse
Hepburn, Richard James (Virginia Tech, 2004-06-14)
Equine gastric glandular mucosal ulceration can have a prevalence of 58%, yet its etiology is poorly understood. In man Helicobacter pylori is the most common cause of gastritis and peptic ulcer disease. Helicobacter is uniquely able to colonize the stomach, via the action of cytoplasmic urease. Different Helicobacter species have been isolated from many mammals but none has yet been cultured from the horse. Three tests used to identify human Helicobacter infection were applied to the horse. Test 1: PCR amplification of Helicobacter specific DNA, n=12. Test 2: the Pyloritek™ rapid urease test (RUT), n=15. Test 3: the 13C-urea blood test, n=8. Gastroscopy and antral biopsy was performed in all horses. All horses demonstrated the presence of Helicobacter specific gene material by PCR. Biopsy specimens from 7/15 horses were urease positive by RUT. Significant 13C enrichment of the body CO2 pool was found in all horses after intragastric administration 13C-urea (p<0.05). As Helicobacter is currently the only known gastric urease positive microorganism, the demonstration of this activity in horses positive by PCR strongly supports the presence of an equine gastric Helicobacter species. Variations of 13C-urea blood test were further examined and a single protocol was found to be most applicable. As the horse is a hind gut fermenter, the effect of cecal urease on the test was examined by laparoscopic intracecal administration of 13C-urea. Significant cecal urease activity was demonstrated however the timing of peak 13C enrichment may limit any effect on the gastric test to 90 minutes onwards.
Local Administration of Botulinum Toxin Type-B in the External Anal Sphincter of Horses Produces Transient Reduction of Peak Anal Pressure
Adam-Castrillo, David (Virginia Tech, 2003-06-27)
Toxins produced by the Gram-positive bacteria Clostridium botulinum cause transient chemodenervation of mammalian muscle. The toxin binds to specific proteins within cholinergic presynaptic nerve terminals which regulate the release of acetylcholine in the synaptic space resulting is loss of muscle activation and function. Local injections with botulinum toxins are currently used in humans for the treatment of disorders that benefit from prolonged neuromuscular blockade such as strabismus, blepharospasm, focal dystonias, spasticity, tremors, and anal fissures. Injections with botulinum toxin type A into the internal or external anal sphincter cause relaxation of the anal canal and allow healing of chronic anal fissures. Perineal lacerations in mares, which occur during foaling often dehisce after surgical repair due to the high pressure across the incision resulting from accumulation of feces in the rectum. We hypothesized local injections of Clostridium botulinum type B toxin into the external anal sphincter could cause a decrease in anal pressures, thus reducing the incidence of dehiscence if used before surgical repair of perineal laceration in mares. The purpose of this project was to determine the effects of BTB injection in the external anal sphincter in normal horses. Our hypothesis was that local injection of BTB would result in transient reduction of anal tone without causing clinical side effects. Peak and resting anal sphincter pressures of horses were measured with a custom made rectal probe connected to a pressure transducer. Pressures were measured before treatment and after injection with Clostridium botulinum type B toxin (BTB) or saline. Dose titration with 500, 1000, 1500 and 2500 units of BTB was completed. The horses' physical changes, behavior, and anal pressure were recorded. Injection of 1000 units of BTB produced significant reduction in peak anal pressure from days 2 to 84 when compared to control animals (P<0.05). Maximal effect of the toxin was observed within the first 15 days after injections followed by a slow return to baseline over 168 days. Injection in the anal sphincter with 2500 units of BTB in one horse produced signs of depression, generalized weakness, and dysphagia for 14 days. Clinical side effects were not observed in horses after injections with 500, 1000, or 1500 units of BTB. In summary, local injections of botulinum toxin type-B in the external anal sphincter of horses caused transient relaxation of the anus and reduction of peak anal pressures. Systemic side effects were observed in one horse, which suggested a narrow dosage range to avoid toxicity. Further research to test the effects of botulinum toxin in clinical cases is needed to determine the full potential of this treatment modality.
Magnetic Resonance Imaging of the Normal Equine Larynx
Henderson, Cortney Erik (Virginia Tech, 2006-06-22)
A study was performed to establish the appearance of normal equine laryngeal cartilages using magnetic resonance imaging. Specimens were acquired from clinically normal horses that were euthanized for reasons other than respiratory disease. Three in situ and 5 ex vivo larynges were imaged using a 0.3 Tesla system. Images were obtained in the transverse plane using T1-weighted 3D spin echo, T2-weighted 3D spin echo, T2-weighted gradient echo, short tau inversion recovery (STIR), and proton density spin echo sequences. Five ex vivo larynges were also imaged in the transverse plane using a 1.5 Tesla system, sequences included T1-weighted 3D spin echo, T2-weighted 3D turbo-spin echo, turbo inversion recovery (TIRM), and proton density spin echo sequences. A frozen gross laryngeal specimen was sliced in 5-mm transverse sections for comparison to the MR images. Excellent correlation was found between MR images and the gross transverse sections. Successful imaging was accomplished using both imaging systems; however, the 1.5 Tesla system yielded superior image resolution. The 0.3 Tesla imaging system would accommodate the intact equine head, which was not possible using the 1.5 Tesla MRI system. The internal morphology of the laryngeal cartilages was clearly identified in all imaging sequences obtained. Cartilages were found to differ in signal intensity based on the tissue composition and imaging sequences performed. MRI was determined to be a useful imaging modality for evaluating the cartilage morphology of the equine larynx. Further investigation is required to document pathologic morphology.
Magnetic resonance imaging-guided Treatment of equine Distal interphalangeal Joint collateral ligaments: 2009–2014
White, Nathaniel A. II; Barrett, Jennifer G. (Frontiers, 2016-09-05)
Objectives: To determine the outcome of treating distal interphalangeal joint collateral ligament (DIJCL) desmopathy using magnetic resonance imaging (MRI)-guided ligament injection. Methods: Medical records of 13 adult horses diagnosed with DIJCL desmopathy using low-field MRI and treated by MRI-guided ligament injection of mesenchymal stem cells and/or platelet-rich plasma (PRP) were reviewed. Information collected included signalment, MRI diagnosis, treatment type, time to resolution of lameness, and level of exercise after treatment. results: Collateral ligament inflammation was diagnosed as a cause of lameness in 13 horses. MRI was used to guide the injection of the injured DIJCL. All lameness attributed to DIJCL desmopathy resolved with the resulting level of performance at expected (10) or less than expected (3). conclusion and clinical relevance: Injection of the DIJCL can be safely completed in horses standing in a low-field magnet guided by MRI as previously demonstrated in cadaver specimens. The positive response in all horses suggests that administration of stem cells or PRP along with rest and appropriate shoeing may be a safe and useful treatment for DIJCL desmopathy.
Methods to Detect Apoptosis in Equine Peripheral Blood Neutrophils from Normal Healthy Adult Horses
Wereszka, Marta (Virginia Tech, 2007-06-15)
Apoptosis is a form of "planned cell death" and is an essential component of normal tissue differentiation and functional regulation. Neutrophil apoptosis facilitates down regulation of the inflammatory response while minimizing "by stander" injury to normal tissue, and disruption of this process by various diseases may have a significant negative impact on patient recovery. Consequently, neutrophil apoptosis has been the focus of research in many species. However, methods for measuring apoptosis have not been evaluated in the horse. The goal of this study was to adapt previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species for use in equine peripheral blood neutrophils. To achieve this goal the experiment was divided into three parts: 1. Induce apoptosis and necrosis in equine peripheral blood neutrophils using previously used known inducers and examine the relationship between exposure time and percentage of affected cells; 2. Measure percentage of apoptosis and necrosis using three methods of detection: a) Annexin-V Fitc PI assay, b) Homogenous caspase 3/7 assay and c) Light microscopy and; 3. Compare the results between the three methods of apoptosis detection to determine if results are comparable The hypothesis was that previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species can be adapted for use in equine peripheral blood neutrophils. Venous blood samples were collected aseptically from the jugular vein of eight horses. Isolation of neutrophils was performed using density gradient centrifugation on percoll. In part 1 of the experiment aliquots of the neutrophil suspension were cultured in the presence of four known inducers of apoptosis; actinomycin D, staurosporin, cycloheximide and sodium hypochlorite, at four different concentrations (table 2). A fifth population was to induce necrosis using a freeze-thaw cycle and bleach. A control sample was examined (no inducer) to determine spontaneous rate of apoptosis. The aliquots were cultured and the percentage of apoptosis determined at two sequential time points for each horse. Apoptosis was measured at either 30 minutes and 3 hours or 6 and 12 hours by three simultaneous methods: (1) annexin-V FITC PI assay (AVF), (2) homogenous caspase assay (HC) and (3) light microscopy (MS). The AVF and HC methods detect events associated with early apoptosis whilst MS detects nuclear changes which are late events of apoptosis. Using AVF and MS apoptotic cells are able to be differentiated from necrotic cells. In part two of the experiment the agreement and reproducibility between AVF and MS was further examined. In this part of the experiment neutrophils were isolated from the peripheral blood of 10 normal healthy adult horses. Each isolated sample was cultured with 80µM Actinomycin D for 12 hours and a control sample (no inducer) also prepared. Three triplicate samples were next set up from both the induced and control sample and apoptosis was determined using both AVF and MS. In part 3 of the experiment, data was analyzed using the mixed model ANOVA following log transformation of the data. Main effects of treatment, concentration and time were analyzed. Statistical significance was considered if P was < 0.05. The relationship between the three techniques; light microscopy, flow cytometry and the fluorescent plate reader, was investigated using Spearman rank correlation coefficients (Fisher's Z transformation). The Bland-Altman approach for method analysis was used to further characterize the correlation between results obtained via light microscopy and flow cytometry. Statistical significance was considered if P < 0.05. All inducers increased the percentage of apoptotic cells at either one or more time point and results were most comparable between AVF and MS. Increasing exposure time increased percentage of apoptotic neutrophils for all inducers using AVF and MS (p<0.0001). For both AVF and MS, cycloheximide and staurosporin induced apoptosis significantly above control levels at 3, 6 and 12 hours; actinomycin D at 6 and 12 hours and bleach at 3 and 6 hours as well was 12 hours for AVF only. With HC induction of apoptosis was detected earlier with bleach at 30 minutes and 3 hours and staurosporin at 30 minutes, 3 and 6 hours. Apoptosis was detected only at 6 hours for cycloheximide. Increasing concentration of inducer significantly increased the percentage apoptotic cells for staurosporin and cycloheximide between the lowest and highest concentration using AVF (p<0.001). For both AVF and MS, increasing concentration of bleach decreased the percentage of apoptotic cells (p<0.05). Increasing the concentration of staurosporin resulted in an increase in apoptosis at 30 minutes and 3 hours. Both bleach and the freeze-thaw cycle induced necrosis at all time periods excluding 30 minutes for the freeze-thaw cycle (p<0.0001). Spearman rank correlation coefficients revealed a very high correlation for percentage apoptosis and necrosis between AVF and MS (r2 = 0.91, 95% CI 0.89 – 0.93). A high correlation was also present for AVF and HC (r2 = 0.75, 95% CI 0.69 – 0.79) and MS and HC (r2 = 0.76, 95% CI 0.71 – 0.81). The lower limit of the confidence intervals suggests there is some concern about the similarity between AVF, HC and MS, HC. The Bland and Altman statistical approach indicates that both AVF and MS are highly reproducible methods with minimal variation between the triplicate samples (AVF: 8.9%, 95% CI 6.25 – 11.6%, MS 7.9%, 95% CI 6 – 9.8%). The mean difference between the two methods is 6.7% (95% CI 3.89 – 9.42%). The 95% limits of agreement indicate that results from MS can be 8.7% below to 22% above results from AVF (95% CI -13.41 – 26.7%). These findings indicate that caspase activation may occur prior to phosphatidylserine externalization and visible nuclear changes, which is in accordance with previously published data. We discovered that actinomycin D induces significant and reproducible equine peripheral blood neutrophil apoptosis in a time dependant fashion. Similarly, necrosis results from a freeze-thaw cycle or high concentration of bleach and is suitable as a positive control for necrosis. Apoptosis was effectively detected using AVF assay and results indicate good correlation between AVF and MS with an acceptably low mean difference. MS could serve as an inexpensive, simple and quick on site method to rapidly verify results attained from AVF. Induction of apoptosis using the HC was not consistent and can not be recommended based on the results of this study. Future investigation aimed at evaluating assays multiplexed to the AVF which detect other aspects of the apoptotic pathway would lead to increased confidence of results and further evidence of the mode of cell death prior to undertaking clinical studies.
Outcomes of Medical Treatment for Pathologies of the Equine Foot Diagnosed with Magnetic Resonance Imaging
Gutierrez-Nibeyro, Santiago Daniel (Virginia Tech, 2008-07-15)
A retrospective study was performed to determine the prevalence of foot pathologies of horses subjected to magnetic resonance imaging for foot lameness and to determine the long-term outcome of horses after medical treatment. The MR studies of 95 horses were interpreted retrospectively by a boarded certified radiologist. Follow-up information was obtained from medical records, owners and referring veterinarians via telephone questionnaires. Long term response to treatment (minimum of 12 months) was recorded. Horses were divided in two different groups based on the diagnosis and on the treatment using intrasynovial antiinflammatory drugs or not. Logistic regression analysis was performed to compare the outcome between the two groups. The null hypothesis was that the proportion of horses treated successfully between treatment protocols was similar. A diagnosis based on magnetic resonance imaging was made in all horses. Approximately 30% of horses had ≥ 4 lesions, which were determined to be responsible for the lameness and 70% of horses had navicular bone abnormalities. Treatment was determined by individual clinician judgment. No significant difference was found in the long-term outcome between treatment groups. This result suggests that intrasynovial antiinflammatory drugs may not provide additional benefit over corrective shoeing, rest followed by controlled exercise in horses with lesions of structures associated with the navicular apparatus or the distal interphalangeal joint.

Browsing by Author "White, Nathaniel A. II"

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