Kayoko Harada

Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells

It has been reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells show multilineage differentiation potential similar to that observed in mesenchymal stem cells. Since DFAT cells can be prepared from a small quantity of adipose tissue, they could facilitate cell-based therapies in small companion animals such as cats. The present study examined whether multipotent DFAT cells can be generated from feline adipose tissue, and the properties of DFAT cells were compared with those of adipose-derived stem cells (ASCs). DFAT cells and ASCs were prepared from the floating mature adipocyte fraction and the stromal vascular fraction, respectively, of collagenase-digested feline omental adipose tissue. Both cell types were evaluated for growth kinetics, colony-forming unit fibroblast (CFU-F) frequency, immunophenotypic properties, and multilineage differentiation potential. DFAT cells and ASCs could be generated from approximately 1g of adipose tissue and were grown and subcultured on laminin-coated dishes. The frequency of CFU-Fs in DFAT cells (35.8%) was significantly higher than that in ASCs (20.8%) at passage 1 (P1). DFAT cells and ASCs displayed similar immunophenotypes (CD44(+), CD90(+), CD105(+), CD14(-), CD34(-) and CD45(-)). Alpha-smooth muscle actin-positive cells were readily detected in ASCs (15.2±7.2%) but were rare in DFAT cells (2.2±3.2%) at P1. Both cell types exhibited adipogenic, osteogenic, chondrogenic, and smooth muscle cell differentiation potential in vitro. In conclusion, feline DFAT cells exhibited similar properties to ASCs but displayed higher CFU-F frequency and greater homogeneity. DFAT cells, like ASCs, may be an attractive source for cell-based therapies in cats.

Mitral valve repair under cardiopulmonary bypass in small-breed dogs: 48 cases (2006-2009)

OBJECTIVE To determine whether mitral valve repair (MVR) under cardiopulmonary bypass would be an effective treatment for mitral regurgitation in small-breed dogs. DESIGN Retrospective case series. ANIMALS 48 small-breed dogs (body weight, 1.88 to 4.65 kg [4.11 to 10.25 lb]; age, 5 to 15 years) with mitral regurgitation that underwent surgery between August 2006 and August 2009. PROCEDURES Cardiopulmonary bypass was performed with a cardiopulmonary bypass circuit. After induction of cardiac arrest, a mitral annuloplasty was performed, and the chordae tendineae were replaced with expanded polytetrafluoroethylene chordal prostheses. After closure of the left atrium and declamping to restart the heart, the thorax was closed. RESULTS Preoperatively, cardiac murmur was grade 3 of 6 to 6 of 6, thoracic radiography showed cardiac enlargement (median vertebral heart size, 12.0 vertebrae; range, 9.5 to 14.5 vertebrae), and echocardiography showed severe mitral regurgitation and left atrial enlargement (median left atrium-to-aortic root ratio, 2.6; range, 1.7 to 4.0). 45 of 48 dogs survived to discharge. Three months after surgery, cardiac murmur grade was reduced to 0/6 to 3/6, and the heart shadow was reduced (median vertebral heart size, 11.1 vertebrae, range, 9.2 to 13.0 vertebrae) on thoracic radiographs. Echocardiography confirmed a marked reduction in mitral regurgitation and left atrium-to-aortic root ratio (median, 1.7; range, 1.0 to 3.0). CONCLUSIONS AND CLINICAL RELEVANCE We successfully performed MVR under cardiopulmonary bypass in small-breed dogs, suggesting this may be an effective surgical treatment for dogs with mitral regurgitation. Mitral valve repair with cardiopulmonary bypass can be beneficial for the treatment of mitral regurgitation in small-breed dogs.

Surgical Closure of an Atrial Septal Defect Using Cardiopulmonary Bypass in a Cat

OBJECTIVE To describe surgical repair of a large atrial septal defect (ASD) in a cat. STUDY DESIGN Clinical report. ANIMAL A 3-year-old, 3.3 kg, intact male Japanese domestic short-haired cat. METHODS A 10.2-mm-diameter ASD detected by echocardiography was surgically corrected because pulmonary vascular resistance-to-systemic vascular resistance ratio (Qp /Qs ) was 3.2. Using cardiopulmonary bypass (CPB), open surgical repair was achieved with an expanded polytetrafluoroethylene (e-PTFE) graft. The priming volume of the CPB circuit was minimized by cutting the CPB tubing, and partially replacing the priming fluid with whole cat blood. To prevent hemodilution associated with use of cardioprotective agents, surgery was performed on the beating heart. RESULTS At 1-year echocardiographic evaluation, the repair was intact, and at 3 years, the cat was alive without need of medication. CONCLUSIONS Large ASD in a cat can be repaired using e-PTFE under CPB.

Comparison of the diuretic effect of furosemide by different methods of administration in healthy dogs

Objective To compare the diuretic effects of subcutaneous (SC) administration of furosemide to conventional methods of administration including intravenous (IV), per os (PO), and constant rate infusion (CRI) in healthy dogs. Design Prospective, randomized, cross-over study. Setting Veterinary university research facility. Animals Seven healthy, adult mongrel dogs (3 males, 4 females). Intervention Each dog in the study was randomly assigned to receive a 2 mg/kg dose of furosemide via a single SC, IV, or PO dose at the beginning (time 0) of an 8-hour study, or via CRI during an 8-hour study period. Urine was collected by emptying the bladder using an indwelling catheter and blood samples were obtained via venipuncture at time 0 for baseline measurements and at 1, 2, 4, 6, and 8 hours into the study. Hourly urine output was calculated in all dogs for each study. Complete blood count, plasma total protein, blood urea nitrogen, creatinine, and renin concentration were measured for each sample. Measurements and Main Results The SC administration of furosemide resulted in a urine output per hour (UOP/h) that peaked at 1 hour with UOP/h returning to baseline at 4 hours after injection. Following IV administration, UOP/h also peaked at 1 hour but returned more rapidly to baseline levels at 2 hours after injection. With PO administration, UOP/h reached a maximum UOP/h at 2 hours but time to return to baseline levels was prolonged to 6 hours after administration. With CRI administration, the time to the maximum UOP/h was delayed to 4 hours after injection but UOP/h was then maintained throughout the study period. Conclusions Total urine output following SC administration of furosemide in healthy dogs was similar when compared to the IV and PO route. Subcutaneous route may be an effective means for administration of furosemide in dogs, particularly when IV access is difficult.OBJECTIVE To compare the diuretic effects of subcutaneous (s.c.) administration of furosemide to conventional methods of administration including intravenous (i.v.), per os (p.o.), and constant rate infusion (CRI) in healthy dogs. DESIGN Prospective, randomized, cross-over study. SETTING Veterinary university research facility. ANIMALS Seven healthy, adult mongrel dogs (3 males, 4 females). INTERVENTION Each dog in the study was randomly assigned to receive a 2 mg/kg dose of furosemide via a single s.c., i.v., or p.o. dose at the beginning (time 0) of an 8-hour study, or via CRI during an 8-hour study period. Urine was collected by emptying the bladder using an indwelling catheter and blood samples were obtained via venipuncture at time 0 for baseline measurements and at 1, 2, 4, 6, and 8 hours into the study. Hourly urine output was calculated in all dogs for each study. Complete blood count, plasma total protein, blood urea nitrogen, creatinine, and renin concentration were measured for each sample. MEASUREMENTS AND MAIN RESULTS The s.c. administration of furosemide resulted in a urine output per hour (UOP/h) that peaked at 1 hour with UOP/h returning to baseline at 4 hours after injection. Following i.v. administration, UOP/h also peaked at 1 hour but returned more rapidly to baseline levels at 2 hours after injection. With p.o. administration, UOP/h reached a maximum UOP/h at 2 hours but time to return to baseline levels was prolonged to 6 hours after administration. With CRI administration, the time to the maximum UOP/h was delayed to 4 hours after injection but UOP/h was then maintained throughout the study period. CONCLUSIONS Total urine output following s.c. administration of furosemide in healthy dogs was similar when compared to the i.v. and p.o. route. Subcutaneous route may be an effective means for administration of furosemide in dogs, particularly when i.v. access is difficult.

Surgical Repair of a Complete Endocardial Cushion Defect in a Dog

OBJECTIVE To describe surgical repair of a complete endocardial cushion defect (ECD) in a dog. STUDY DESIGN Clinical report. ANIMAL A 5-month-old, 9.2 kg male Shetland sheepdog. METHODS Echocardiographic examination revealed an ostium primum atrial septal defect (ASD), an inlet ventricular septal defect (VSD), mitral regurgitation (MR) and tricuspid regurgitation (TR), and a complete ECD was diagnosed. Surgical correction was performed using cardiopulmonary bypass (CPB) via right atriotomy. A polytetrafluoroethylene (PTFE) patch was secured along the margin of the inlet VSD using simple continuous suture, then the cleft in the septal mitral leaflet was sutured. Similarly, the cleft in the septal tricuspid leaflets was sutured. To complete inlet VSD closure, the VSD patch was secured to these sutured leaflets by simple continuous suture. Another PTFE patch was used to close the ostium primum ASD. RESULT After surgery, MR, TR, and interventricular shunting were decreased. The dog was alive 6 years and 5 months after the surgery with no evidence of an interventricular shunt, TR, or other clinical signs. CONCLUSIONS Complete ECD in a dog was corrected using a 2-patch technique under CPB.

Estimating glomerular filtration rate in healthy dogs using inulin without urine collection

The goals of this study were to determine if the glomerular filtration rate (GFR) in dogs could be estimated by plasma inulin clearance and/or infusion inulin clearance analyses without urine collection, and to compare these results with GFR values obtained by urinary inulin clearance analysis. The dogs included in this study were healthy 20 beagles. Inulin clearance values were obtained by urinary inulin clearance, infusion inulin clearance, and plasma inulin clearance techniques. Urinary inulin clearance was 4.09±0.52 ml min(-1) kg(-1) (body weight); infusion inulin clearance, 4.01±0.49 ml min(-1) kg(-1); and plasma inulin clearance, 4.14±0.66 ml min(-1) kg(-1). The urinary inulin clearance was strongly correlated with infusion inulin clearance and weakly correlated with plasma inulin clearance. The GFR for dogs can be estimated by infusion and plasma inulin clearance analyses by blood sampling alone, without urine collection.

Plasma cytokine levels in dogs undergoing cardiopulmonary bypass

UNLABELLED Currently, there are no reports of inflammatory responses to CPB in dogs. We investigated the time course of pro- and anti-inflammatory cytokine levels during and after CPB. ANIMALS The study group included 11 dogs that underwent mitral valve repair with CPB, and the control group included 7 healthy dogs that underwent ovariohysterectomy. METHODS Blood samples from the study group dogs were collected before, during and after surgery and analyzed for plasma levels of interleukin-6 (IL-6), tissue necrosis factor-α (TNF-α), interleukin-10 (IL-10), white blood cells (WBC), and C-reactive protein (CRP). Each inflammatory parameter was also compared with that of the control group dogs. RESULTS After CPB, plasma levels of IL-6, WBC counts, and CRP levels were significantly higher than preoperative levels, and IL-6 levels in the study group were significantly higher than those in the control group. CONCLUSIONS CPB induces a systemic inflammatory response in dogs.

Surgical treatment of severe pulmonic stenosis under cardiopulmonary bypass in small dogs

OBJECTIVES The aim of this study was to report the long-term outcome of the surgical palliation of pulmonic stenosis in dogs. METHODS The subjects comprised three female and six male dogs, mean (±sd) age: 23 (±25) months, mean (±sd) weight: 3·4 (±2·1) kg, diagnosed with severe pulmonic stenosis and right ventricular hypertrophy, with an average preoperative pressure gradient of 153 (±43) mmHg on echocardiography. RESULTS The pressure overload with severe pulmonic stenosis was reduced by valvotomy, i.e., open pulmonary valve commissurotomy, with/without biomembrane patch grafting, under cardiopulmonary bypass. The postoperative pressure gradient at 1 to 7 days was significantly decreased to 65 (±39) mmHg (P<0·05). The reduced pressure gradient was maintained at 58 (±38) mmHg at final follow-up. CLINICAL SIGNIFICANCE Open valvotomy, pulmonary valve commissurotomy and biomembrane patch grafting were effective in reducing obstruction in severe pulmonic stenosis in dogs.

Pathologic Manifestations on Surgical Biopsy and Their Correlation with Clinical Indices in Dogs with Degenerative Mitral Valve Disease.

Background Evaluation of myocardial function is clinically challenging in dogs with degenerative mitral valve disease (DMVD). Although myocardial dysfunction is caused by pathologic degeneration, histopathologic progression is poorly understood. Objectives To characterize myocardial and pulmonary pathologic changes according to severity in dogs with naturally occurring DMVD, and to investigate whether or not pathologic degeneration is reflected by traditional clinical indices. Animals One hundred and seventeen dogs with naturally occurring DMVD. Methods Prospective observational study. Biopsied left atrium (LA), left ventricle (LV), and lung were evaluated histologically, and an attempt was made to correlate pathologic findings with clinical indices. Results Severe myocardial changes were observed in all International Small Animal Cardiac Health Council classes. In the lung, heart failure cell levels were significantly increased in class III patients (P < .0001). In a paired comparison, the LA showed significantly more severe degeneration than the LV, including myocardial fatty replacement, immune cell infiltration, and interstitial fibrosis (P < .0001). In contrast, myocardial cells were more hypertrophied in the LV than in the LA (P < .0001). Left ventricular end‐diastolic dimension (LVEDd) was associated with fatty replacement (P = .033, R 2 = 0.584) and myocardial vacuolization (P = .003, R 2 = 0.588) in the LA. Conclusions and Clinical Importance In DMVD, although severe pathologic changes may be evident even in early stages, there may be pathologic discrepancy between the LA and the LV. Myocardial degeneration may be reflected by clinical indices such as LVEDd and EF.

Post-mortem evaluation of expanded polytetrafluoroethylene (ePTFE) used in mitral valve repair in dogs

Mitral valve repair is one of the treatment options for mitral regurgitation. Expanded polytetrafluoroethylene (ePTFE) is a polymer that has been widely used in cardiovascular surgery. In this case series, we report the autopsy and histological findings in 6 dogs that underwent cardiopulmonary bypass for mitral annuloplasty using ePTFE sheets and chordoplasty using ePTFE sutures. From May 2005 to October 2009, 3 female and 3 male dogs with severe mitral regurgitation underwent mitral valve repair. This case series included 3 Cavalier King Charles spaniels, 2 Maltese, and 1 Shih Tzu. The survival period after surgery was 19-72 (35 ± 19) months. In all the cases, autopsy revealed that the ePTFE sheets and sutures were not damaged and well integrated into the surrounding highly differentiated, connective tissues. Low-power microscopy revealed that in all cases, the tissues surrounding the ePTFE sheet in the mitral valve annulus had almost completely been covered by granulation tissue. No inflammatory infiltrate or thrombogenesis was observed around the ePTFE in any of the cases. There was no evidence of reactive changes in the region surrounding the ePTFE. These results suggest that ePTFE has excellent tissue compatibility and durability and can be effectively used for canine mitral valve repair.