Veterinary Clinical Sciences - Theses
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ItemIsocitrate dehydrogenase 1 R132H: epidermal growth factor receptor and Ki-67 expression in canine gliomas( 2019)The diagnosis, histological classification and grading of canine gliomas can be challenging, particularly in the antemortem. In humans, it is essential to accurately diagnose gliomas as the treatment and prognosis vary between tumour histologic type and grade. Numerous biomarkers have been investigated to aid in the diagnosis and classification of human gliomas, including epidermal growth factor receptor (EGFR), Ki-67 and a point mutation in the isocitrate dehydrogenase 1 gene at codon 132 (IDH1 R132H). Only limited studies looking at these biomarkers have been performed in dogs, from which clear conclusion cannot be drawn. The prognosis for canine glial tumours is considered guarded to poor, with the current treatment options having limited success. Consequently, there is a lack of gold standard treatment for canine glioma. In humans, gliomas are being investigated at a molecular level to identify therapeutic targets and to improve responsiveness to treatment. Numerous molecular targets have been identified including EGFR and IDH1 R132H. Canine gliomas share many morphologic, histologic and immunohistochemical characteristics with human gliomas, suggesting a translational approach to the classification, treatment and prognostication of canine gliomas may be possible. The objectives of this research were: (1) to describe the signalment, presenting clinical signs, diagnostic findings, treatment and survival time of a series of dogs with glial tumours, (2) to investigate the series of canine gliomas for the IDH1 R132H mutation, and for EGFR and Ki-67 expression, utilising immunohistochemistry and (3) to determine if immunoreactivity is associated with tumour histologic type and grade. The clinical data of thirty-one dogs with histologically confirmed glial tumours was reviewed retrospectively. The formalin-fixed paraffin-embedded tumour specimens were evaluated for IDH1 R132H, pan-IDH1 (IDH1 wild-type and mutated IDH1), EGFR and Ki-67 expression. IDH1 R132H and pan-IDH1 expression were recorded as positive or negative. EGFR immunoreactivity was evaluated using a semi-quantitative score, and Ki-67 expression was expressed as the Ki-67 labelling index (LI). Patient signalment, clinical presenting signs and diagnostic investigation findings were similar to previous reports. The IDH1 R132H point mutation was not identified in any (0%) of the canine glial tumours, while all 31 (100%) tumours were positive for pan-IDH1 expression. EGFR expression was identified in 16/31 (51.6%) tumours and expression was significantly greater in high grade gliomas when compared to low grade tumours (P = 0.04). Furthermore, EGFR expression was significantly greater in gliomatosis cerebri when compared to oligodendroglioma (P = 0.002), astrocytoma (P = 0.01), and oligoastrocytoma (P = 0.04). Ki-67 expression was identified in 28/31 (90.3%) gliomas, and the Ki-67 LI was significantly higher in high grade tumours (P = 0.02). A significant moderate correlation between the Ki-67 LI and EGFR immunoreactivity (r = 0.47, P = 0.007) was identified. The IDH1 R132H point mutation was not identified in this series of canine gliomas, and may not be an appropriate biomarker to aid in the classification and treatment of these tumours. EGFR, however, may be a suitable therapeutic target, particularly for gliomatosis cerebri. The Ki-67 LI may aid in the development of a grading scheme for canine gliomas.
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ItemChronic enteropathy in dogs: the role of macrophages and preliminary results in inflammatory cytokines( 2019)Chronic enteropathy (CE) is an umbrella term used in dogs to describe a group of diseases with different aetiologies, characterised by chronic gastrointestinal signs. These diseases are clinically classified according to treatment response as food-responsive (FRE), antibiotic-responsive (ARE), and immunosuppressant-responsive enteropathies (IRE). The first part of this PhD thesis prospectively describes the features of CE that are commonly seen in dogs presenting to a referral centre in Australia; information that has not been available previously. We found that similar to other countries, most dogs with CE are food-responsive, followed by antibiotic-responsive with a minority of immunosuppressant-responsive. Furthermore, our study raised concerns about prolonged antibiotic treatment for dogs with ARE. Firstly, most of these dogs do not respond to treatment completely for prolonged periods (as opposed to dogs with FRE that do), raising the question about the real benefit of antibiotic treatment. Secondly, half of the dogs with ARE required long-term or pulse treatment with antibiotics, which raises concerns about development of bacterial resistance. Our findings highlight the need to find alternative treatment for dogs with ARE in view of the poor long-term outcome. Although most dogs with FRE had long-term remission, adequate dietary trials were not performed until reaching the referral setting. This indicates that better education of general veterinary practitioners about the importance of performing adequate diet trial is needed to improve early disease remission in these dogs. The next focus of the research was to evaluate the role of macrophages in CE; this was achieved by using two macrophage markers: calprotectin and cluster of differentiation 163 (CD163) in immunohistochemical examination. Both immunohistochemical markers highlighted two different populations of macrophages in our intestinal biopsy specimens. Overall the number of CD163 positive cells was higher than calprotectin positive cells both in crypts and villi. Dogs with FRE and IRE had a decreased CD163:calprotectin ratio compared to healthy dogs with an increase in the ratio after treatment. Our results suggest that there is an imbalance in macrophage populations in dogs with FRE and IRE, with partial resolution following clinical response characterised by an increase in the ratio CD163:calprotectin. Interestingly, dogs with ARE not only have a poor long-term response, but also have different macrophage populations from dogs with FRE and IRE; and in fact, are very similar to healthy dogs without change in their macrophage populations with treatment response. These results suggest that macrophages play a role in the pathogenesis of FRE and IRE dogs with normalisation of macrophage populations with treatment response. The CD163 receptor is cleaved during macrophage activation and is released into the circulation as a soluble form. In view of the decreased number of CD163 cells in the intestine of dogs with FRE and IRE at diagnosis (and subsequent increase with clinical remission), we wanted to determine if soluble CD163 could be detected in dog serum, and therefore potentially serve as a biomarker. Two different ELISAs were tested and although one of them showed some signal, further testing of the antibodies used in the assay did not support that the signal was specific for CD163. With this experiment, we were not able to quantify soluble CD163 in dogs, but this molecule retains potential as a biomarker for diagnostic and monitoring purposes in CE as well as in other diseases characterised by macrophage activation. Biomarkers of systemic inflammation were also assessed in the same cohort of dogs and we showed that serum IL-6 decreased in dogs with CE after resolution of clinical signs. Similarly to soluble CD163, cytokines might play a role in further differentiating between the different causes of CE for prognostic and therapeutic purposes. Future studies are needed to assess these cytokines in a larger cohort of dogs to be able to study differences between FRE, ARE, and IRE, and further assess their role as biomarkers. Finally, we studied cytokine production by lymphocytes or monocytes in the peripheral blood of healthy dogs, and the effect of different immunosuppressive treatments on cytokine production. Differential activation of lymphocytes or monocytes can easily be achieved by using specific activators in whole blood. The advantage of this technique is that there is minimal handling of the cells with less risk of iatrogenic activation. Cytokine production was affected by cyclosporine and prednisolone, but not by mycophenolate, leflunomide, or azathioprine. Cyclosporine inhibited production of tumour necrosis factor (TNF), interferon gamma and IL-10 by lymphocytes whereas prednisolone inhibited TNF production by both lymphocytes and monocytes. Our findings suggest that this methodology can be used to monitor dogs treated with both drugs concurrently – although this needs to be further assessed with future studies. Future studies highlighted by our research suggest more in-depth assessment of serum cytokines as biomarkers for dogs with CE not only for monitoring purposes, but to determine if different patterns of cytokines can be useful to refine the classification of CE. Similarly, whole blood stimulation can be used to better assess underlying priming of the immune system and to monitor treatment response. Finally, our findings suggest that macrophages play a significant role in the pathophysiology of CE in dogs, particularly in FRE and IRE, but additional work is required to better understand their function in CE.
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ItemAssociations between the radiographic appearance of vascular channels in proximal sesamoid bones, their microstructural characteristics and past racing performance in thoroughbreds( 2019)Reasons for performing the study: ‘Sesamoiditis’ is classically defined by equine practitioners as the presence of and abnormalities in vascular channel appearance within the proximal sesamoid bones (PSB). It is the most common finding in Thoroughbred yearling presale radiographs, as well as often being evaluated on radiographs of adult racehorses with lameness and poor performance. Despite this, the pathogenesis and clinical significance of changes in vascular channel morphology are poorly understood, and the association of ‘sesamoiditis’ with racing performance is inconsistently reported. Objectives: To determine the microstructural characteristics of the PSB associated with the radiographic appearance of vascular channels in Thoroughbred racehorses using micro-computed tomography (µCT), and to determine whether the presence, number and size of vascular channels has an association with past racing performance. Methods: Study design was cross-sectional. One pair of PSB were isolated from a randomly selected forelimb of 59 Thoroughbred racehorses presenting for post-mortem examination over the study period. Each PSB (n=118) was radiographed, assigned a vascular channel grade using previously published and novel radiographic grading systems, then imaged using µCT and similarly assessed. Racing history for each horse was collected. Uni- and multi-variable generalized linear models accounting for clustering at the horse level were generated to investigate associations between radiographic, µCT and performance variables. Results: All PSB had vascular channels observed on µCT originating from the abaxial border (mean 3.6, s.d 0.89), yet in only 63.6% (75/118) were channels observed radiographically. PSB with a higher bone volume fraction (OR 1.08; P=0.031) and wider channel diameter on µCT (OR 20.67; P=0.001) were more likely to have vascular channels identified on radiographs. Radiographic channel number (OR 0.96; P=0.043) and channel size (OR 0.96; P=0.049) were negatively associated with career placings. Main Limitations: Only the forelimb proximal sesamoid bones were collected, radiographs of isolated bones avoided normal superimposition of soft tissue encountered in the live horse, and a cross-sectional study design meant changes in sesamoid vasculature over time and work load could not be assessed. Conclusions: The ability to identify vascular channels radiographically indicates widening of channels and densification of the bone. Increased radiographic channel number and size is associated with poorer measures of past performance suggesting that these changes are not desirable.
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ItemInflammation and endothelial perturbation in canine abdominal surgery: the potential modulatory effect of lidocaine( 2019)Complication rates following emergency laparotomy surgery are high, with organ dysfunction being a commonly encountered post-operative complication. Given the endothelium acts as the interface between the systemic circulation and the organs, its function is vital to maintaining organ health. The endothelium is in a constant state of flux, impacted largely by the local environment of which it is a part. In the presence of wide-spread systemic inflammation, inflammatory mediators precipitate change to the structure of the endothelial glycocalyx. These changes result in shedding of the endothelial glycocalyx and alteration of the endothelial phenotype. The endothelium may, as a result, lose the capacity to regulate vasomotor tone, and shift toward a pro-inflammatory and pro-coagulant state. This predisposes to reduced tissue oxygen delivery, and organ dysfunction may ensue. This thesis aimed to answer two key questions: does surgical trauma induced in canine patients undergoing emergent abdominal surgery invoke a systemic inflammatory response and subsequent endothelial activation? And if so, does lidocaine, a proposed immunomodulatory drug, mitigate this effect when given in the post-operative period? Chapter two provides a detailed review of endothelial structure and function, and current literature pertaining to systemic inflammation and endothelial activation in the context of abdominal surgery. Chapter two also examines the literature regarding the proposed mechanisms through which lidocaine acts as an immunomodulatory drug, and reviews publications that investigate the use of lidocaine as an anti-inflammatory drug in human patients after abdominal surgery. Chapter three is a randomized, blinded clinical trial quantifying the effect of emergency abdominal surgery on the concentration of markers of systemic inflammation and endothelial perturbation in canine patients in the post-operative period. The trial also assessed the potential use of lidocaine as a post-operative immunomodulatory therapy in dogs having undergone laparotomy. Fifty canine patients undergoing abdominal surgery were enrolled in the study. Patients were randomized into two separate groups: a study group receiving lidocaine intravenously, and a control group receiving 0.9% NaCl intravenously for a twelve-hour period following abdominal surgery. Blood samples were gathered prior to surgery, followed by six and twelve hours post-operatively. Concentrations of markers of systemic inflammation (IL-6) and markers of endothelial perturbation (VEGF and HA) were quantified via means of ELISA at each time point. Results revealed a significant increase in the concentration of markers of systemic inflammation and endothelial perturbation in post-operative blood samples. No immunomodulatory or endothelial preserving effect of lidocaine was appreciated.
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ItemUpdate on clinicopathological assessment of renal health in non-racing greyhounds( 2019)Background: Serum creatinine concentrations differ in greyhounds compared with non-sighthounds, but it is not known whether urine creatinine concentrations also differ and whether any difference would influence the interpretation of the urine protein to creatinine ratio (UPC). Additionally, there is some evidence for greyhounds having higher serum symmetric dimethylarginine (SDMA) than non-sighthounds, but this has yet to be confirmed in healthy non-racing greyhounds. Objectives: The objectives of this study were fourfold: (1) to compare the urine creatinine concentrations in healthy greyhounds and a control group of healthy non-sighthounds, (2) to determine the UPC reference interval in healthy greyhounds and to compare this with the UPC reference interval in a control group of healthy non-sighthounds, (3) to determine the serum SDMA concentration reference interval in healthy greyhounds and to compare this with the serum SDMA concentration reference interval in a control group of healthy non-sighthounds and with a previously established canine serum SDMA concentration reference interval, and (4) to establish whether lean body mass is correlated with serum creatinine and urine creatinine concentrations in greyhounds. Methods: The study used an observational cross-sectional design and included 98 clinically healthy non-racing greyhounds and 24 non-sighthound dogs with similar weight, age and sex distributions, as determined by t-test and chi-squared tests. SDMA, urine creatinine concentration and UPC values were measured from blood and urine samples. Linear regression was used to compare the greyhound and non-sighthound groups. Greyhound reference intervals were determined for SDMA and UPC using non-parametric methods. These were compared with the reference intervals for the non-sighthound group and with current International Renal Interest Society guidelines. In the greyhound sample, the association of urine creatinine with thigh circumference, height and weight was estimated using Pearson correlation. Statistical significance was set at P < 0.05 for all analyses. Results: Mean urine creatinine was approximately 22% higher in greyhounds than non-sighthounds after adjusting for urine concentration (P < 0.05). The upper limit of the greyhound UPC reference interval was 0.20 or 0.42, depending on whether strict or moderate exclusion criteria, respectively, were applied. The mean UPC was 29% lower in greyhounds than non-sighthounds, but this difference was not statistically significant (P = 0.1). The serum SDMA reference interval for greyhounds was 6.3–19.7 µg/dL (0.31–0.98 µmol/L). The upper end of this interval was higher than the upper limit of the published canine reference interval (6–13 µg/dL), and the mean concentration was statistically significantly higher in greyhounds (13.0 µg/dL) than non-sighthounds (10.2 µg/dL, P < 0.001). In greyhounds, there were weak correlations between the three morphometric measurements and both serum creatinine and urine creatinine after adjusting for urine concentration. Conclusions and clinical importance: These findings provide further evidence that greyhounds require several breed-specific reference intervals when evaluating renal function. Apart from having higher serum creatinine, greyhounds also have higher SDMA and higher urine creatinine when compared to non-sighthounds. Although UPC trended slightly lower in greyhounds, this finding was not significant, and therefore the threshold for non-proteinuria set by IRIS guidelines appears to be appropriate for greyhounds based on the calculated reference interval.