Andrew E. Kyles

Human tissue-engineered blood vessels for adult arterial revascularization.

There is a crucial need for alternatives to native vein or artery for vascular surgery. The clinical efficacy of synthetic, allogeneic or xenogeneic vessels has been limited by thrombosis, rejection, chronic inflammation and poor mechanical properties. Using adult human fibroblasts extracted from skin biopsies harvested from individuals with advanced cardiovascular disease, we constructed tissue-engineered blood vessels (TEBVs) that serve as arterial bypass grafts in long-term animal models. These TEBVs have mechanical properties similar to human blood vessels, without relying upon synthetic or exogenous scaffolding. The TEBVs are antithrombogenic and mechanically stable for 8 months in vivo. Histological analysis showed complete tissue integration and formation of vasa vasorum. The endothelium was confluent and positive for von Willebrand factor. A smooth muscle–specific α-actin–positive cell population developed within the TEBV, suggesting regeneration of a vascular media. Electron microscopy showed an endothelial basement membrane, elastogenesis and a complex collagen network. These results indicate that a completely biological and clinically relevant TEBV can be assembled exclusively from an individuals own cells.

Comparison of transdermal fentanyl and intramuscular oxymorphone on post-operative behaviour after ovariohysterectomy in dogs

The effects of transdermal fentanyl and i.m. oxymorphone on behavioural and physiological responses, after ovariohysterectomy in dogs, were investigated. The study involved three groups of 10 dogs: fentanyl/surgery (FS), oxymorphone/surgery (OS), fentanyl/control (FC). A transdermal fentanyl delivery system (50 microg hour(-1)) (FS and FC) was applied 20 hours before surgery, or i.m. oxymorphone (OS) was administered. After ovariohysterectomy (FS and OS) or anaesthesia alone (FC), dogs were continuously videotaped for 24 hours and a standardised hourly interaction with a handler performed. The videotapes were analysed, and interactive and non-interactive behaviours evaluated. In addition, pain and sedation scores, pulse and respiratory rates, rectal temperature, arterial blood pressure, plasma cortisol and plasma fentanyl concentrations were measured. This study showed that transdermal fentanyl and i.m. oxymorphone (0.05 mg kg(-1)) produced comparable analgesic effects over a 24 hour recording period. I.m. oxymorphone produced significantly more sedation and lower rectal temperatures than transdermal fentanyl. There were no significant differences between groups in respiratory and heart rates, and arterial blood pressures.

Indicators of Malignancy of Canine Adrenocortical Tumors: Histopathology and Proliferation Index

Tumors of the adrenal cortex account for 10–20% of the naturally occurring Cushings syndrome diagnosed in dogs. Differentiating between adrenocortical adenoma and carcinomas is often difficult. The purposes of this study were to determine which histopathologic criteria can be used as markers for malignancy in canine adrenocortical tumors and the relevance of the proliferation marker, Ki-67, for differentiation between cortical adenomas and carcinomas. Twenty-six adrenocortical carcinomas, 23 adenomas, and 11 normal adrenal glands were examined. Morphologic criteria significantly associated with adrenocortical carcinomas included a size larger than 2 cm in diameter, peripheral fibrosis, capsular invasion, trabecular growth pattern, hemorrhage, necrosis, and single-cell necrosis, whereas hematopoiesis, fibrin thombi, and cytoplasmic vacuolation were significantly associated with adrenocortical adenomas. The mean (± SD) proliferation index, measured by immunohistochemistry for the Ki-67 antigen, was 9.3 ± 6.3% in carcinomas, 0.76 ± 0.83% in adenomas, and 0.58 ± 0.57% in normal adrenal glands. The Ki-67 proliferation index was significantly higher in carcinomas compared with adenomas and normal adrenal glands. A threshold value of the proliferation index of 2.4% reliably separated carcinomas from adenomas. Based on these results, it appears that thorough evaluation of morphologic features combined with immunohistochemical assessment of the proliferation index is extremely useful for differentiating between adrenocortical adenomas and carcinomas in dogs.

Dried solidified blood calculi in the urinary tract of cats.

We have noted an increased number of calculi submitted to the Gerald V. Ling Urinary Stone Analysis Laboratory, University of California, Davis, that do not contain crystalline material but appear to be composed of dried solidified blood (DSB). Canine and feline laboratory records from 1986-2003 were reviewed for samples composed of >99% DSB. No calculi from dogs were found, but specimens from 49 cats were composed of >99% DSB, of which almost half (n = 22) had been submitted after 2001. The DSB calculi had been removed surgically or by postmortem examination from all areas of the upper and lower urinary tract. All samples were well formed and could be divided in half with Rongeur forceps. Detailed case information was available for 12 cats. Urinalyses were available for 9 of the 12 cats, and the mean specific gravity was 1.017 (range, 1.009-1.032). Red blood cells were reported in the sediment of all cats, with most containing >100 RBC/hpf. Ureteral obstructions but no radio-opaque calculi were visible by radiography, including radiographic contrast studies. Reports of ultrasound examinations were available for 10 cats, and discrete calculi were not recorded. In addition to polarized light microscopy, infrared spectroscopy and electron probe microanalysis were performed on a subset (n = 6, DSB calculi; n = 3, control calculi) of samples. Significantly more carbon, nitrogen, and sulfur (P = .012, P = .02, and P = .012, respectively) were present in the elemental analysis of the DSB calculi than in the control calculi, suggesting that the DSB calculi are primarily formed from organic material. At this time, we are uncertain why these DSB calculi become solidified, and we recommend that samples be submitted both in formalin and preservative free to further investigate their etiology.

Immunosuppression with a combination of the leflunomide analog, FK778, and microemulsified cyclosporine for renal transplantation in mongrel dogs.

Background. The leflunomide analog, FK778, is a selective pyrimidine synthesis inhibitor. In rodent models, FK778 is efficacious in the prevention of allograft and xenograft rejection, and a combination of FK778 and cyclosporine has synergistic immunosuppressive efficacy. Methods. Heterotopic renal transplantation was performed in 20 dogs. Dogs were randomly assigned to three treatment groups: Neoral alone (n=6), FK778 alone (n=7), or a combination of Neoral and FK778 (n=7). Dogs were killed when the plasma creatinine concentration exceeded 7 mg/dL. A complete postmortem examination was performed and the type of acute–active allograft rejection described. Results. A combination of Neoral and FK778 significantly prolonged allograft survival (P =0.0007), with median survival times of 14.5 days for Neoral alone, 7 days for FK778 alone, and 36 days for Neoral and FK778. Allograft histologic changes were consistent with acute–active allograft rejection in 19 of 20 dogs: in the Neoral-alone group, four dogs were type IB, and two were type IIA; in the FK778-alone group, five dogs were type IB, and one was type IIB; and in the Neoral and FK778 group, three dogs were type IB, three were IIA, and one dog was type III. The dog with type III rejection appeared to experience acute sepsis before rejection. Vomiting, diarrhea, and histologic gastritis and enteritis were commonly observed in dogs treated with the combination of Neoral and FK778. Conclusions. A combination of Neoral and FK778 prolonged allograft survival in a robust rejection model. Further investigation of FK778 in organ transplantation is warranted.

Adrenalectomy and Caval Thrombectomy in a Cat With Primary Hyperaldosteronism

A 13-year-old, castrated male, domestic longhaired cat was diagnosed with primary hyperaldosteronism from an adrenal gland tumor and a thrombus in the caudal vena cava. Clinical signs included cervical ventriflexion, lethargy, weakness, inappetence, and diarrhea. Laboratory tests revealed hypokalemia, normonatremia, hyperglycemia, hypophosphatemia, and elevated creatine kinase activity. Hypokalemia worsened despite oral potassium supplementation. An adrenalectomy and caval thrombectomy were successfully performed utilizing deliberate hypothermia followed by progressive rewarming.

Computed tomographic lymphography of the thoracic duct by mesenteric lymph node injection.

OBJECTIVE To document a novel technique to image the thoracic duct and its tributaries by contrast enhanced computed tomography (CT) lymphography. STUDY DESIGN Clinical report. ANIMALS Dogs (n=6) idiopathic chylothorax. METHODS Ultrasonography was used to guide percutaneous injection of intestinal lymph nodes with nonionic iodinated contrast medium for preoperative CT lymphography of the thoracic duct in 6 dogs with chylothorax. Thoracic CT images were acquired immediately after contrast medium injection. All dogs had subtotal pericardectomy and thoracic duct ligation. Postoperative thoracic duct lymphography was performed in 3 dogs. Superficial cervical lymph node lymphography was performed in 2 dogs to determine cervical lymphatic contribution to thoracic effusions. RESULTS Preoperative thoracic duct lymphography using this technique was successful in delineating the cisterna chyli, thoracic duct, and associated lymphatic vessels in all dogs. Immediate postoperative lymphography performed in 2 dogs revealed successful duct ligation in 1 dog and persistent lymphatic leakage in the other. A 1-month postoperative thoracic duct lymphogram performed in 1 dog revealed unsuccessful ligation or recannulation of 1 of 3 redundant vessels seen preoperatively. CONCLUSION Percutaneous CT lymphography results in excellent detection of the thoracic duct and abnormal thoracic duct drainage patterns both pre- and postoperatively. The contribution of superficial cervical lymph node drainage to reoccurrence of effusions can be evaluated. CLINICAL RELEVANCE Percutaneous CT lymphography using ultrasound-guided contrast medium injection should be considered as an alternative to conventional open abdominal approaches to radiographic or CT lymphography.

Use of Rheolytic Thrombectomy in the Treatment of Feline Distal Aortic Thromboembolism

The purpose of this prospective clinical trial was to evaluate the safety and efficacy of a commercially available rheolytic thrombectomy system in the treatment of naturally occurring feline aortic thromboembolic disease. All 6 cats enrolled in the investigation were affected at the level of the distal aorta and had signs of the disease affecting both pelvic limbs. Cats were anesthetized and an arteriotomy was performed on 1 carotid artery to gain access to the arterial system. Selective arterial angiography was used to confirm the presence of thromboembolic disease. The thrombectomy system was advanced to the level of the thrombus using fluoroscopic guidance. Repeat angiography was used intermittently to assess progress of thromboembolus dissolution throughout the procedure. The use of the rheolytic thrombectomy system resulted in successful thrombus dissolution in 5 of 6 cats. Three of 6 cats survived to discharge. Both of these results compare favorably with conventional therapies used in the treatment of this disease. Feline distal aortic thromboembolism is a frustrating disease that warrants a guarded to poor prognosis. Rheolytic thrombectomy may provide veterinarians with an alternative therapy in the treatment of thromboembolic diseases, including feline distal aortic thromboembolism.

Compared with cyclosporine, ISATX247 significantly prolongs renal-allograft survival in a nonhuman primate model

Background. ISATX247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first study to compare the survival times of renal allografts in nonhuman primates treated with either ISATX247 or cyclosporine. Methods. Adult, male cynomolgus monkeys were divided into blood-group compatible and mixed-lymphocyte, stimulation-mismatched, donor-recipient pairs. Heterotopic renal transplantation and bilateral native nephrectomies were performed. The monkeys were placed into either an ISATX247 or cyclosporine treatment group. Both groups were dosed twice daily to maintain a 12-hour drug-trough level of 150 ng/mL. Whole-blood concentrations of ISATX247 and cyclosporine, complete blood counts, and serum chemistry profiles were performed three times a week. Euthanasia was performed if the serum creatinine concentration became 7 or more mg/dL or a serious complication developed. Results. The group receiving ISATX247 (n=8) survived significantly (P=0.0036) longer than the group receiving cyclosporine (n=7). The mean trough blood concentration of ISATX247 was 120±32 ng/mL and cyclosporine was 189±130 ng/mL. The average area under the curve0–12 for ISATX247 was 6045±1679 ng/mL/hr and for cyclosporine was 4919±823 ng/mL/hr. The average calcineurin inhibition at trough blood concentrations was 80±11% for ISATX247 and 48±12% for cyclosporine. Conclusions. Allografts in monkeys treated with ISATX247 survived significantly longer than those treated with cyclosporine. On the basis of survival times and degree of calcineurin inhibition, ISATX247 is a more potent immunosuppressive agent than cyclosporine in this nonhuman primate model of renal-allograft transplantation.

Fluvastatin in combination with RAD significantly reduces graft vascular disease in rat cardiac allografts

Background. RAD is a potent immunosuppressive agent that has been shown to be effective in preventing acute and chronic allograft rejection in animal models. The HMGCoA reductase inhibitors have been found to reduce the incidence of graft vascular disease (GVD) in heart transplant patients and in animal models. This study was designed to investigate the effects of fluvastatin or pravastatin in a rodent model of GVD produced using low doses of RAD to prevent acute rejection. Methods. Hearts from Fisher 344 rats were heterotopically transplanted to Lewis rat recipients. RAD was administered orally at 0.5 mg/kg per day for days 0 to 14 and then 0.25 mg/kg per day for an additional 85 days to prevent acute rejection but allow for the development of GVD. Pravastatin (20 mg/kg per day) or fluvastatin (2 or 6 mg/kg per day) was added to the RAD treatment. At the end of a 100-day treatment period, the hearts were harvested for morphometric and histopathologic examinations. Results. Rats treated with fluvastatin, at either dose, had a significant (P ≤0.0239) decrease in coronary arterial intimal thickening (GVD) of approximately 43%. Rats treated with pravastatin had a 22% reduction in GVD that did not reach statistical significance. Treatment with fluvastatin, but not pravastatin, decreased the degree of endomyocardial mononuclear cell infiltration seen with RAD administered alone. Conclusions. Fluvastatin significantly decreased GVD in a rat model produced using low-dose RAD immunosuppression. To a lesser extent, pravastatin also decreased GVD in this model. These data lend further support for the study of fluvastatin, pravastatin, and other HMG-CoA reductase inhibitors for the prevention of GVD in cardiac transplant patients.