Development and Evaluation of a Tissue-Engineered Fibrin-based Canine Mitral Valve Three-dimensional Cell Culture System.

Myxomatous mitral valve disease is the most common cardiac disease of the dog, but examination of the associated cellular and molecular events has relied on the use of cadaveric valve tissue, in which functional studies cannot be undertaken. The aim of this study was to develop a three-dimensional (3D) cell co-culture model as an experimental platform to examine disease pathogenesis. Mitral valve interstitial (VIC) and endothelial (VEC) cells were cultured from normal and diseased canine (VIC only) valves. VICs were embedded in a fibrin-based hydrogel matrix and one surface was lined with VECs. The 3D static cultures (constructs) were examined qualitatively and semiquantitatively by light microscopy, immunofluorescence microscopy and protein immunoblotting. Some constructs were manipulated and the endothelium damaged, and the response examined. The construct gross morphology and histology demonstrated native tissue-like features and comparable expression patterns of cellular (α-smooth muscle actin [SMA] and embryonic smooth muscle myosin heavy chain [SMemb]) and extracellular matrix associated markers (matrix metalloproteinase [MMP]-1 and MMP-3), reminiscent of diseased valves. There were no differences between constructs containing normal valve VICs and VECs (type 1) and those containing diseased valve VICs and normal valve VECs (type 2). Mechanical manipulation and endothelial damage (type 3) tended to decrease α-SMA and SMemb expression, suggesting reversal of VIC activation, but with retention of SMemb+ cells adjacent to the wounded endothelium consistent with response to injury. Fibrin-based 3D mitral valve constructs can be produced using primary cell cultures derived from canine mitral valves, and show a phenotype reminiscent of diseased valves. The constructs demonstrate a response to endothelial damage indicating their utility as experimental platforms.

Culture and characterisation of canine mitral valve interstitial and endothelial cells.

Valve interstitial cells (VICs) have an important role in the aetiopathogenesis of myxomatous mitral valve disease (MMVD) in the dog. Furthermore, there is evidence that valve endothelial cells (VECs) also contribute to disease development. In addition to examining native valve tissue to understand MMVD, another strategy is to separately examine VIC and VEC biology under in vitro culture conditions. The aim of this study was to isolate and characterise canine mitral VICs and VECs from normal dog valves using a combination of morphology, immunohistochemistry and reverse transcription PCR (RT-PCR). Canine mitral VECs and VICs were isolated and cultured in vitro. The two cell populations exhibited different morphologies and growth patterns. VECs, but not VICs, expressed the endothelial markers, platelet endothelial cell adhesion molecule (PECAM-1 or CD31) and acetylated low density lipoprotein (Dil-Ac-LDL). Both VECs and VICs expressed vimentin and embryonic non-smooth muscle myosin heavy chain (SMemb), an activated mesenchymal cell marker. The myofibroblast marker, alpha smooth muscle actin (α-SMA), was detected at the mRNA level in both VEC and VIC cultures, but only at the protein level in VIC cultures. The morphological heterogeneity and expression of non-endothelial phenotypic markers in VEC cultures suggested that a mixture of cell types was present, which might be due to cell contamination and/or endothelial-mesenchymal transition (EndoMT). The use of a specific endothelial culture medium for primary VEC cultures enhanced the endothelial properties of the cells and reduced α-SMA and SMemb expression.

Cell maceration scanning electron microscopy and computer-derived porosity measurements in assessment of connective tissue microstructure changes in the canine myxomatous mitral valve.

Canine myxomatous mitral valve disease is associated with changes in the valve extracellular matrix (ECM). The aim of this study was to examine the use of cell macerated scanning electron microscopy (CMSEM) in evaluating ECM changes in a small sample of valves and to quantify these changes using computer-aided image analysis of sample porosity (a measure of structural disorganisation and collagen loss). The distinct layered structure of the de-cellularised matrix could be seen in the normal valve and there were marked changes in layers and ECM organisation as the disease progressed. Clearly visible and quantifiable, statistically significant changes were found in valve porosity across the entire leaflet thickness and particularly in the valve mid and distal zones. All of these changes are presumed to affect the mechanical function of the valve. In conclusion, CMSEM with computed image analysis can be used to visualise and measure tissue structural changes in a quasi-3-dimensional manner in normal and diseased tissues.

Efficacy of pimobendan in the prevention of congestive heart failure or sudden death in Doberman Pinschers with preclinical dilated cardiomyopathy (the PROTECT Study).

BACKGROUND: The benefit of pimobendan in delaying the progression of preclinical dilated cardiomyopathy (DCM) in Dobermans is not reported. HYPOTHESIS: That chronic oral administration of pimobendan to Dobermans with preclinical DCM will delay the onset of CHF or sudden death and improve survival. ANIMALS: Seventy-six client-owned Dobermans recruited at 10 centers in the UK and North America. METHODS: The trial was a randomized, blinded, placebo-controlled, parallel group multicenter study. Dogs were allocated in a 1:1 ratio to receive pimobendan (Vetmedin capsules) or visually identical placebo. The composite primary endpoint was prospectively defined as either onset of CHF or sudden death. Time to death from all causes was a secondary endpoint. RESULTS: The proportion of dogs reaching the primary endpoint was not significantly different between groups (P = .1). The median time to the primary endpoint (onset of CHF or sudden death) was significantly longer in the pimobendan (718 days, IQR 441-1152 days) versus the placebo group (441 days, IQR 151-641 days) (log-rank P = 0.0088). The median survival time was significantly longer in the pimobendan (623 days, IQR 491-1531 days) versus the placebo group (466 days, IQR 236-710 days) (log-rank P = .034). CONCLUSION AND CLINICAL IMPORTANCE: The administration of pimobendan to Dobermans with preclinical DCM prolongs the time to the onset of clinical signs and extends survival. Treatment of dogs in the preclinical phase of this common cardiovascular disorder with pimobendan can lead to improved outcome.

An unusual morphology of patent ductus arteriosus in a dog.

A 12-week old, entire female Border terrier weighing 3·5 kg was presented for investigation of a continuous left heart base murmur. The clinical presentation and preoperative echocardiogram were consistent with a standard morphology of patent ductus arteriosus (PDA) but a discrete ductal vessel was not identified during surgical dissection. Surgery had to be abandoned due to deterioration of the patient's condition under general anaesthesia which led to cardiorespiratory arrest and death despite attempts at resuscitation. Necropsy identified a recess within the wall of the aorta communicating with the pulmonary artery via an ostium at the heart base which determined this structure as an intramural PDA. This morphology of PDA is previously unreported. This report demonstrates that an intramural PDA is not readily identifiable surgically because of the absence of a discrete ductal vessel and it is important to appreciate that unusual morphologies of PDA may occur.

Canine tissue-specific expression of multiple small leucine rich proteoglycans.

Small leucine rich proteoglycans (SLRPs) are important constituents of extracellular matrix (ECM) and contribute to the production, organization and remodelling of collagen and elastin through complex biological systems. The relative expression and distribution of SLRPs in a variety of different mammalian tissues is poorly characterized. The aim of this study was to map the expression of seven SLRPs (biglycan, versican, prolargin, fibromodulin, osteoglycin, decorin and lumican) in seven tissues (bone, cartilage, cruciate ligament, skin, ventricular myocardium, mitral valve and cornea) in young adult dogs using a combination of quantitative real-time PCR, immunohistochemistry and protein immunoblotting. Clear and consistent patterns of SLRP expression and distribution were identified for the seven tissues examined, with the greatest SLRP expression in cartilage, skin, cornea and mitral valve, and the least expression in myocardium. In general, lumican and prolargin had the greatest expression across the seven tissues whilst osteoglycin was the least abundantly expressed SLRP. These data provide a SLRP profile for different canine tissues which can inform future studies of SLRP expression in development and disease.

Outcome in 55 dogs with pulmonic stenosis that did not undergo balloon valvuloplasty or surgery.

OBJECTIVE: To determine the outcome, independent predictors of cardiac death, and the Doppler-derived pressure gradient cut-off for predicting cardiac death in dogs with pulmonic stenosis, with or without tricuspid regurgitation, that do not undergo balloon valvuloplasty or valve surgery. METHODS: Review of medical records of two UK referral centres between July 1997 and October 2008 for all cases of pulmonic stenosis that had no balloon valvuloplasty or valve surgery. Inclusion criteria included a diagnosis of pulmonic stenosis; spectral Doppler pulmonic velocity greater than 1·6 m/s; characteristic valve leaflet morphological abnormalities. Exclusion criteria included concurrent significant cardiac defects, including tricuspid dysplasia. Dogs with tricuspid regurgitation were included. Dogs were classified according to Doppler-derived pressure gradients into mild, moderate or severe pulmonic stenosis categories. RESULTS: Presence of tricuspid regurgitation and severe stenosis were independent predictors of cardiac death. A pulmonic pressure gradient of more than 60 mmHg was associated with 86% sensitivity, and 71% specificity of predicting cardiac death. CLINICAL SIGNIFICANCE: There is an increased probability of cardiac death in those cases which have a pulmonary pressure gradient greater than 60 mmHg and tricuspid regurgitation, though the effect of severity of tricuspid regurgitation on outcome was not measurable because of small sample sizes. These animals might benefit from intervention.

Efficacy and safet of pimobendan in canine heart failure caused by myxomatous mitral valve disease.

OBJECTIVES: To evaluate the clinical efficacy and safety of pimobendan by comparing it with ramipril over a six-month period in dogs with mild to moderate heart failure (HF) caused by myxomatous mitral valve disease (MMVD). METHODS: This was a prospective randomised, single-blind, parallel-group trial. Client-owned dogs (n = 43) with mild to moderate HF caused by MMVD were randomly assigned to one of two groups, which received either pimobendan (P dogs) or ramipril (R dogs) for six months. The outcome measures studied were: adverse HF outcome, defined as failure to complete the trial as a direct consequence of HF; maximum furosemide dose (mg/kg/day) administered during the study period; and any requirement for additional visits to the clinic as a direct consequence of HF. RESULTS: Treatment with pimobendan was well tolerated compared with treatment with ramipril. P dogs were 25 per cent as likely as R dogs to have an adverse HF outcome (odds ratio 4.09, 95 per cent confidence interval 1.03 to 16.3, P = 0.046). CLINICAL SIGNIFICANCE: R dogs had a higher overall score and thus may have had more advanced disease than P dogs at baseline (P = 0.04). These results should be interpreted cautiously but such a high odds ratio warrants further investigation.

Long-term follow-up of dogs with patent ductus arteriosus.

Postocclusion survival data from dogs with left-to-right shunting patent ductus arteriosus (PDA) was available from 80 dogs, diagnosed from 1990 to 2000. Of these, 37 had undergone a procedure to close the ductus and were re-evaluated at the time of this study; clinical data from the follow-up examination was compared with that from the original examination. Radiographically, the right ventricle remained apparently enlarged, and the aortic bulge associated with dilation of the descending aorta did not disappear after closure. On M-mode echocardiography, left ventricular chamber diameter in diastole and systole and left ventricular posterior wall in systole decreased significantly. Mitral endocardiosis was a common feature. Residual flow was evident in 46 per cent of the animals. Late closure occurred in 8 per cent of the dogs, and trivial recanalisation in 19 per cent. The maximum survival time postclosure was 168 months and, after non-occlusion, 114 months, suggesting that dogs with PDA follow an unpredictable course. However, there was a significant difference in survival times between the corrected and non-corrected group.

Review of left-to-right shunting patent ductus arteriosus and short term outcome in 98 dogs.

The case records of 98 dogs with a left-to-right shunting patent ductus arteriosus (PDA) were reviewed. There were 35 breeds represented, with a female to male ratio of 3:1. Forty per cent of the dogs were older than one year at initial presentation and 31 per cent had clinical signs attributable to PDA. A left heart base continuous murmur of grade IV/VI or higher was noted in 90 per cent of the dogs. On electrocardiography, the most common abnormalities were tall R waves (63 per cent) and deep QII waves (62 per cent). The radiographic triad of dilation of the descending aorta with enlargement of the main pulmonary artery segment and left atrium, typical of PDA, was noted in only 26 per cent of cases. Two-dimensional (2D) and M-mode echocardiography detected left atrial enlargement (35 per cent) and an increased left ventricular diameter in diastole (82 per cent) and systole (84 per cent) as the most common abnormalities. Doppler echocardiography demonstrated increased aortic outflow velocities in 66 per cent of cases. The overall short-term successful outcome in this study was 95 per cent. There was no significant difference between surgical ductal ligation using a standard technique or the Jackson-Henderson technique in terms of survival, occurrence of haemorrhage or residual shunting. The number of interventional procedures used in this study was too low for statistical comparison, but there appeared to be a trend towards a higher rate of residual shunting and a lower fatality rate using a coil occlusion technique.

Retrieval of a patent ductus arteriosus coil following embolisation to the right subclavian artery.

A two-month-old female Tibetan terrier was re-presented with an incomplete closure of the patent ductus arteriosus. Following a second attempt to close the shunt by coil embolisation, arterial embolisation occurred. The successful removal of the embolised coil is described.

Myasthenia gravis associated with cutaneous lymphoma in a dog.

A middle-aged golden retriever presenting with exercise intolerance and multifocal cutaneous nodules was diagnosed as having non-epitheliotropic cutaneous lymphoma with concurrent myasthenia gravis. Treatment with corticosteroids induced short-term remission of the lymphoma and alleviation of myasthenic signs.

Analysis of murmur intensity, duration and frequency components in dogs with aortic stenosis.

A study was undertaken to investigate the relationship between murmur intensity, murmur duration, duration to peak intensity and frequency components with degree of aortic stenosis in boxers. Measurements were made from phonocardiograms obtained from 35 boxers with ejection-type murmurs, and values were compared with those obtained for aortic flow velocity measured by Doppler echocardiography. Murmur intensity graded by auscultation was significantly correlated with aortic flow velocity (P < 0.001), and murmur duration, expressed as a percentage of systole was significantly correlated with aortic flow velocity (P < 0.001), independent of heart rate. Dogs with early systolic murmurs not exceeding 50 per cent of systole had aortic flow velocities of less than 1.5 m/second and no echocardiographic abnormalities, in contrast with dogs with murmurs of longer duration. Dogs with only high frequency components had lower aortic velocities than those that also had components in the medium frequency range (P < 0.01).

Treatment of Trombicula autumnalis infestation in dogs and cats with a 0.25 per cent fipronil pump spray.

To evaluate the efficacy of fipronil in controlling trombiculid infestations, 18 dogs and three cats infested with Trombicula autumnalis larvae were treated monthly from examination to the end of the Trombicula season (range one to four months) with a 0.25 per cent fipronil spray applied to the whole body, with particular emphasis on the feet, face, ears, perineum and tail. No other antiparasite measures were used. Follow-up was by clinical examination and telephone interview until the end of the Trombicula season (range one to four months). No adverse effects were seen. Monthly treatment controlled trombiculids in 15 dogs. In two dogs localised pedal reinfestations were controlled with additional local application of fipronil to the feet every 14 days. In one dog therapy was of no benefit. In the three cats, treatment was initially effective, but generalised infestations recurred after seven to 10 days. Fipronil is a safe and effective treatment for trombiculid infestations in dogs. Residual activity lasts for 14 to 30 days. Further studies are required to examine the apparent short duration of efficacy in cats.

Pulmonary venous flow characteristics as assessed by transthoracic pulsed Doppler echocardiography in normal dogs.

Transthoracic Doppler echocardiography was used to evaluate the technique of measuring and normal patterns of pulmonary venous flow in fourteen normal dogs. Polyphasic pulmonary venous flow profiles were obtained in all dogs, consisting of one (S) or two (SE and SL) systolic forward flow waves, one early diastolic forward flow wave (D), one reverse flow wave (R) related to atrial contraction, and one reverse flow wave (R2) observed after cessation of systolic flow. Pulmonary venous flow was laminar in 9 dogs (65%). Maximal flow velocity during systole (0.39 +/- 0.14 m/sec) was significantly lower (P < 0.01) than in early diastole (0.56 +/- 0.14 m/sec). During late diastole peak flow velocity was 0.20 +/- 0.08 m/sec and maximum R2 velocity was 0.17 +/- 0.05 m/sec. Duration of mitral A-wave was significantly greater (P < 0.05) than R-wave duration in all dogs (0.075 +/- 0.010 vs 0.058 +/- 0.012 sec). These results can be used for comparison with patterns found in disease states.

Uraemic myoclonus: an example of reticular reflex myoclonus?

Two patients are described who developed action, reflex myoclonus during acute renal failure. In both cases the myoclonus was abolished after the intravenous administration of clonazepam. We suggest that the characteristic action myoclonus, which occurs in both acute renal failure and postanoxic encephalopathy, is caused by a disturbance of function in the lower brainstem reticular formation.