Quantitative cDNA-AFLP analysis for genome-wide expression studies.

An improved cDNA-AFLP method for genome-wide expression analysis has been developed. We demonstrate that this method is an efficient tool for quantitative transcript profiling and a valid alternative to microarrays. Unique transcript tags, generated from reverse-transcribed messenger RNA by restriction enzymes, were screened through a series of selective PCR amplifications. Based on in silico analysis, an enzyme combination was chosen that ensures that at least 60% of all the mRNAs were represented by an informative sequence tag. The sensitivity and specificity of the method allows one to detect poorly expressed genes and distinguish between homologous sequences. Accurate gene expression profiles were determined by quantitative analysis of band intensities, and subtle differences in transcriptional activity were revealed. A detailed screen for cell cycle-modulated genes in tobacco demonstrates the usefulness of the technology for genome-wide expression analysis.

Mechanical thrombolysis of venous thrombosis in an animal model with use of temporary caval filtration.

PURPOSE: To test the effect of temporary caval filtration on pulmonary emboli when a mechanical thrombolytic device is used to treat venous thrombosis and to test the effects of a modified device on caval patency at 30-day follow-up. MATERIALS AND METHODS: In a canine model of iliocaval subacute thrombosis, mechanical thrombolysis was performed with use of an 8-F over-the-wire Arrow-Trerotola Percutaneous Thrombolytic Device (PTD) with a 9-mm (iliac) or 15-mm (inferior vena cava [IVC]) basket. In six procedures, the device was made of nitinol monofilament, and in another six, it was made of braided stainless steel. All procedures were performed with a nitinol expandable sheath (temporary filter) in the suprarenal IVC. Low-molecular-weight heparin was given daily after the procedure. Venography, pulmonary arteriography, measurement of blood gases, and pulmonary artery (PA) pressure measurement were performed before and after the procedure and at 30-day follow-up. Pulmonary arteriograms from the group treated with stainless-steel devices were compared to those from an earlier group of animals in which the identical procedure was performed without caval filtration. The IVC was examined histologically. RESULTS: Thrombolysis was successful in all animals. Rare segmental and subsegmental pulmonary emboli (PE) were seen arteriographically; compared to procedures without filters, there was a significant reduction in PE (P <.002). However, a mild increase in pulmonary artery pressure, decrease in pH, and increase in pCO(2) were observed postprocedurally. At 30-day follow-up (n = 11), IVC patency was preserved in 45% (n = 5) of animals overall. Caval patency was significantly better in animals in which the combination of stainless-steel devices was used (five of six = 83% vs zero with nitinol device; P =.015). Histologically, the stainless-steel device caused little intimal injury and fibrosis-less than that seen with the nitinol device. CONCLUSIONS: Temporary filtration reduces, but does not completely eliminate, PE during mechanical thrombolysis. The stainless-steel device results in less intimal injury and better caval patency than the nitinol device.

The porcine hemodialysis access model.

PURPOSE: To create a porcine hemodialysis access model that reliably reproduces intimal hyperplasia (IH) of the outflow vein similar to that which causes access failure in human patients undergoing dialysis treatments. MATERIALS AND METHODS: Surgical technique for creation of side-to-side iliac-artery-to-ipsilateral-iliac-vein (IAV) native fistulas and IAV conduits was optimized in three standard-bred pigs. Persistent patency of fistulas and conduits was demonstrated in two additional pigs allowed to survive for 1 week. IAV fistulas and contralateral 2-cm polytetrafluoroethylene IAV conduits were created in five additional pigs. Venous outflow from these fistulas and conduits was evaluated with venography and intravascular ultrasound (IVUS) immediately after creation (day 0) and at 2-week intervals for as long as 64 days. Animals were killed at 30 days (n = 1), 42 days (n = 2), or 64 days (n = 2), and the arteries, veins, and conduits were evaluated histologically. RESULTS: IAV native fistulas remained patent until the animals' death and conduits remained patent for at least 14 days in four of five pigs; both the fistula and conduit likely occluded before 16-day follow-up in the fifth pig. At 42-64 days, venography demonstrated maximum fistula outflow vein diameter stenoses of 53%-76% and maximum conduit outflow vein stenoses of 44%-84%, and IVUS demonstrated maximum area stenoses of 64%-86% and 43%-82%, respectively. Three of five conduits occluded, one before 16-day follow-up, one between 14 and 28 days, and the other after 42 days. Histologic sections demonstrated IH predominantly affecting the veins at the anastomoses and central (cephalad) to the anastomoses in all pigs. CONCLUSION: This porcine model reproduces IH in the fistula or conduit outflow vein with measurable stenosis. Such a model might allow relevant preclinical evaluation of interventional devices and techniques intended to reduce the effects of IH in human patients undergoing dialysis treatments.

Suprarenal intraarterial infusion of alloxan and streptozotocin during balloon occlusion of the juxtarenal abdominal aorta: a simple technique for inducing diabetes mellitus in canines with reduced mortality.

RATIONALE AND OBJECTIVES: The authors performed this study to evaluate the mortality and morbidity associated with a simple technique for inducing diabetes in dogs--suprarenal intraarterial infusion of alloxan and streptozotocin during balloon occlusion of the juxtarenal abdominal aorta. MATERIALS AND METHODS: The authors attempted to induce diabetes in six purpose-bred dogs. After the dogs were fasted for 12 hours, the abdominal aorta at the level of the origin of the renal arteries was occluded with an angioplasty balloon introduced by means of a femoral approach. A 3-F microcatheter (n = 1) or infusion wire (n = 5) was introduced via the percutaneous transluminal angioplasty catheter and positioned at the level of the celiac axis, and a mixture of streptozotocin (20-25 mg/kg) and alloxan (20-25 mg/kg) was infused. Diabetes was considered to have been induced if the dogs experienced sustained hyperglycemia. RESULTS: There were no deaths during the follow-up period (range, 7 months to 2 1/2 years). A diabetes-like state was induced in five of the six dogs, and no nephrotoxicity was seen. Diabetes was not induced in one dog owing to caudal migration of an undersized balloon during the infusion; this also resulted in reversible renal damage. CONCLUSION: This simple technique is effective for inducing diabetes in dogs, and morbidity and mortality rates are lower than those reported in the literature with other described techniques.

Short-term patency and safety of an expanded polytetrafluoroethylene encapsulated endoluminal device at the venous anastomosis of a canine arteriovenous graft model.

PURPOSE: To determine the safety and short-term patency of a polytetrafluoroethylene (PTFE)-encapsulated carbon-lined endoluminal device (ED) deployed across the venous anastomosis of arteriovenous conduits. MATERIALS AND METHODS: Arteriovenous grafts (n = 16) were created between femoral arteries and veins in eight female canines and allowed to mature 30 days +/- 5 (SD). Five were excluded before implantation because of thrombosis or intragraft stenosis. Deployment was conducted in the remaining 11 anastomoses. Fistulography and intravascular ultrasound (IVUS) were performed before and after the procedure and 1 month postimplantation. Stent migration, apposition, and stenosis were evaluated. The angle of the anastomosis was compared before and after deployment and at follow-up. Mural thrombus thickness, percentage of surface covering, and percentage of endothelialization within the device were measured histologically. RESULTS: There was no significant migration. By explant, all devices were completely apposed. Stenosis occurred in three of nine grafts. The angle of the venous anastomosis decreased by 29.5 degrees (posteroanterior) and 32.4 degrees (oblique) after ED deployment. There was a further decrease of 6.1 degrees (posteroanterior) and 3.2 degrees (oblique) during the 4-week follow-up period. Hemostasis was difficult to achieve in this animal model. Five required more than 1 hour to achieve hemostasis manually. Six in three animals were closed with a Perclose device, achieving immediate hemostasis; however, three (one in each animal) re-bled intermittently 2 weeks after implantation for an average of 9.3 days. The puncture site of each graft that bled was radiographically shown abnormal. CONCLUSION: The ED can be deployed without stent migration and is completely apposed and patent after 4 weeks. Although bleeding was a problem with this animal model, delayed bleeding complications associated with puncture site abnormalities were seen only in grafts closed with a percutaneous suturing device.

Leafy gall formation is controlled by fasR, an AraC-type regulatory gene in Rhodococcus fascians.

Rhodococcus fascians can interact with many plant species and induce the formation of either leafy galls or fasciations. To provoke symptoms, R. fascians strain D188 requires pathogenicity genes that are located on a linear plasmid, pFiD188. The fas genes are essential for virulence and constitute an operon that encodes, among other functions, a cytokinin synthase gene. Expression of the fas genes is induced by extracts of infected plant tissue only. We have isolated an AraC-type regulatory gene, fasR, located on pFiD188, which is indispensable for pathogenesis and for fas gene expression. The combined results of our experiments show that in vitro expression of the fas genes in a defined medium is strictly regulated and that several environmental factors (pH, carbon and nitrogen sources, phosphate and oxygen content, and cell density) and regulatory proteins are involved. We further show that expression of the fas genes is controlled at both the transcriptional and the translational levels. The complex expression pattern probably reflects the necessity of integrating a multitude of signals and underlines the importance of the fas operon in the pathogenicity of R. fascians.

Percutaneous transmyocardial intracardiac retroperfusion shunts: technical feasibility in a canine model.

PURPOSE: To test the technical feasibility of creating a left ventricle to coronary sinus shunt using endovascular techniques. MATERIALS AND METHODS: By means of a right jugular vein approach, a needle puncture was made from the coronary sinus to the left ventricle in 10 dogs. The tracts were balloon dilated and lined with 6-mm Wallstents. Shunt patencies, immediate and 4-hours later, were fluoroscopically assessed by contrast material injection into the left ventricle. Blood pressure, pulse, oxygen saturation, and cardiac rhythm were monitored. The dogs were then euthanized. Thoracic cavities and hearts were dissected and inspected. RESULTS: Technical success and immediate shunt patency were 100%. No cardiac dysrhythmias, electrocardiographic changes, or reduction in voltage potential were seen. Eight (80%) of the shunts were patent at 4 hours, one (10%) had thrombosed, and one dog died. Nine (90%) dogs had no pericardial hematoma and one (10%) had minimal pericardial blood from needle passes into the pericardial sac. The coronary sinuses were intact and no injuries to the valve leaflets or chordae tendineae were seen. The puncture sites were from the coronary sinus, 1-2 mm (mean, 1.3) from its auricular orifice, into the left ventricle, just below the inferior margin of the posterior leaflet of the mitral valve. One dog died at 3 hours with no preceding electrocardiographic evidence of impending demise. Autopsy showed no pericardial hematoma and the heart findings were no different from the other nine dogs. CONCLUSIONS: Creation of a left ventricle to coronary sinus shunt with use of endovascular techniques is technically feasible. Study of a transmyocardial intracardiac coronary retroperfusion shunt to deliver oxygenated blood to the ischemic myocardium is warranted.

Local delivery of nadroparin via hydrogel-coated angioplasty balloon: effect on platelet deposition and smooth muscle cell proliferation--an experimental study.

PURPOSE: To assess the feasibility of intravascular delivery of nadroparin, a low-molecular-weight heparin, via hydrogel-coated angioplasty balloons, and the effects of nadroparin delivered in this manner on platelet deposition and smooth muscle cell (SMC) proliferation. MATERIALS AND METHODS: Tritiated nadroparin was used to determine the nadroparin-carrying capacity of the hydrogel-coating, kinetics of release from the balloons, and, in four pigs, delivery of the nadroparin to the iliac arterial wall. Platelet deposition in nadroparin-treated iliac arteries versus contralateral iliac arteries dilated with saline-loaded, hydrogel-coated balloons was quantified in seven pigs using 111Indium-labeled platelets. Smooth muscle cell proliferation in nadroparin and saline-treated iliac arteries in 10 pigs was evaluated 7 days after angioplasty with use of proliferating cell nuclear antigen. RESULTS: Approximately 98 international units of nadroparin were delivered by the hydrogel-coated balloon, the majority to the angioplasty site and distal vessel. There was a trend toward decreased platelet deposition in nadroparin-treated arteries, but statistical significance was not achieved (P = .1563). Medial SMC proliferation was decreased in the nadroparin-treated arteries in nine of 10 pigs (P = .0137). CONCLUSIONS: Hydrogel-coated balloons may be used to deliver nadroparin to the arterial wall, with measurable levels of the drug delivered to the site of angioplasty, and with resultant decrease in SMC proliferation.

Brachytherapy for the prevention of stenosis in a canine hemodialysis graft model: preliminary observations.

PURPOSE: To determine whether gamma brachytherapy can prevent in-stent stenosis in hemodialysis grafts. MATERIALS AND METHODS: Six-millimeter polytetrafluoroethylene arteriovenous grafts were created bilaterally in six dogs. After 1 month, Wallstents spanning the venous anastomosis were placed to accelerate restenosis. Gamma irradiation (12 Gy) was delivered endoluminally to one of the two grafts by using an iridium 192 source; thus, each animal served as its own control. Fistulography was performed monthly for 10 months or until graft thrombosis, with measurement of stenosis at each time point. At the conclusion of the study period, the treated area was examined histologically, and a computer model was used to calculate the volume of intimal hyperplasia. RESULTS: Delayed stent migration resulted in exclusion of one dog. In the remaining five dogs; maximum stenosis across all time intervals was less for the treated side (P < .04), and the volume of intimal hyperplasia was less for the treated side (P < .045). In one animal studied at 1 year, this trend reversed in terms of percentage stenosis but not total neointimal volume. CONCLUSION: Brachytherapy with 192Ir (gamma) delivered at the time of stent placement reduces restenosis in this hemodialysis graft model, but, depending on the parameter evaluated (stenosis vs total volume of neointima), the benefit may wane or even reverse with time.

Technique for construction of an in vivo model of simulated pulmonary metastases.

RATIONALE AND OBJECTIVES: Existing methods used to simulate pulmonary metastases are unsatisfactory. The aim of this study was to create a simple in vivo model of pulmonary metastases by endobronchial deployment of small high-density beads in anesthetized dogs. METHODS: Commercially available decorative beads measuring 2 and 4 mm in diameter and of high density (600 to 1200 Hounsfield units) were deployed in the peripheral airways of anesthetized dogs using catheter and guide wire manipulations through an endotracheal tube. RESULTS: A total of 65 beads were placed in five dogs. Computed tomography demonstrated that 41 (63%) were satisfactorily located in the lung periphery, 9 (14%) were unsatisfactorily located in large airways, and 15 (23%) were not visible. CONCLUSIONS: The endobronchial deployment of small high-density beads in the peripheral airways of anesthetized dogs is a novel and effective technique for creation of an in vivo model of pulmonary metastases.

Preclinical in vivo testing of a rotational mechanical thrombolytic device.

PURPOSE: To establish the safety and efficacy of the Arrow Trerotola mechanical percutaneous thrombolytic device (PTD) for restoring patency of thrombosed hemodialysis grafts. MATERIALS AND METHODS: The hindlimb model of dialysis grafts was created in six dogs. Animals had either unilateral (n = 4) or bilateral (n = 2) polytetrafluoroethylene grafts, totaling eight grafts. Grafts were deliberately clotted 48 hours before thrombolysis. Thrombolysis was performed with five different versions of the PTD constructed of stainless steel (n = 12) or nitinol (n = 26) and rotated with use of a hand-held motor drive. After thrombolysis, fistulography was performed. RESULTS: Thirty-eight procedures were performed with the PTD, with 100% success. Thirty-day patency, evaluated in a subset of 15 procedures, was 100%. Complications included a single arterial embolus (2.6%) and eight device breakages (21%, all but two with the stainless steel version); none had any clinical consequences. A final modification of the nitinol device yielded 11 consecutive procedures without further breakage. No residual thrombus occurred in any procedure. Pathologic examination showed no significant injury to the vessels or neointima. CONCLUSION: The PTD is highly effective for mechanical thrombolysis in an animal model of clotted dialysis grafts. Based on this animal model, the device appears safe in its final modified form.

Pulmonary emboli from pulse-spray and mechanical thrombolysis: evaluation with an animal dialysis-graft model.

PURPOSE: To compare pulmonary emboli resulting from pulse-spray pharmacomechanical thrombolysis (PSPMT) and mechanical thrombolysis performed to declot dialysis-access grafts. MATERIALS AND METHODS: Polytetrafluoroethylene arteriovenous shunts were created in eight dogs and were deliberately clotted at monthly intervals. Animals were randomly assigned to treatment with pulse-spray urokinase thrombolysis or a low-speed rotational percutaneous thrombolytic device. Perfusion imaging, pulmonary-artery pressure measurements, and pulmonary arteriography were performed before and after each procedure. RESULTS: A total of 22 procedures were performed (11 PSPMT and 11 mechanical thrombolysis). Declotting was successful in all procedures, with 100% 30-day patency. Segmental defects were seen on perfusion images after 10 (91%) of 11 PSPMT procedures and two (18%) of 11 mechanical thrombolysis procedures (P < .002). Transient increases in pulmonary-artery pressure occurred in the PSPMT group. Complete resolution of emboli and return to baseline pressures were seen in all cases, even after multiple (up to four) procedures in the same animal. There was no histologic evidence of pulmonary infarction in either group. CONCLUSION: The percutaneous thrombolytic device is effective for declotting dialysis grafts in dogs and results in statistically significantly fewer pulmonary emboli compared with PSPMT.

Percutaneous creation of arteriovenous hemodialysis grafts: work in progress.

PURPOSE: To develop a technique of percutaneous creation and/or revision of arteriovenous hemodialysis access grafts, and to study the feasibility of the technique in the acute setting. MATERIALS AND METHODS: Arteriovenous shunts, consisting of silicone-covered Wallstents 6 (n = 10) or 8 (n = 1) mm in diameter and 11-17 cm in length, were created in 10 mongrel dogs. All shunts were created percutaneously except one, in which the carotid artery was exposed surgically and the jugular vein accessed percutaneously to create a straight external graft. All other grafts were femoral loops (femoral artery to femoral vein). Eight were tunneled subcutaneously and three were left external. All shunts were delivered through 10-F peel-away sheaths. RESULTS: The 11 shunts were allowed to flow for a mean of 4 hours (range, 1.0-6.5 hours) before the animals were killed. Leakage around the "anastomoses" was not observed. Leaks in the silicone covering were observed, resulting in one early shunt failure. Two attempted survival experiments were aborted due to early shunt dislodgment at 1.5 and 3.5 hours. CONCLUSIONS: Percutaneous arteriovenous shunt creation is feasible through acceptably small sheaths. Peel-away sheath placement of the stent circumvents the technical problem of placing a closed device into both artery and vein. This technique may allow percutaneous revision of existing hemodialysis access grafts and possibly de novo creation of grafts in humans, but modification of the grafts will be needed to prevent dislodgment.

Volume detection threshold: quantitative comparison of computed radiography and screen-film radiography in detection of pneumothoraces in an animal model that simulates the neonate.

PURPOSE: To quantitatively compare computed radiography (CR) and screen-film radiography (SFR) in the detection of pneumothorax in an animal model that simulates the neonate. MATERIALS AND METHODS: Three rabbits underwent placement of 5-F catheters in the right pleural space. Eight CR and eight SFR images were obtained. Volume-controlled pneumothorax was induced by incrementally injecting 4 cm3 of air (24 cm3 total) and four CR and four SFR images were obtained after each injection. Four radiologists blindly viewed the images twice (eight readings per image, 1,600 total observations). A dichotomous yes-no score for the presence of pneumothorax was used to calculate the volume detection threshold (VDT), defined as the air volume at which 50% of the pneumothoraces can be detected. The authors plotted the likelihood of a pneumothorax against the air volume by using the confidence indicator. RESULTS: Intraobserver VDT values for CR and SFR were not significantly different. CONCLUSION: CR is as accurate as SFR in the detection of pneumothorax in this quantitative animal model.

Rabbit pneumothorax. Feasibility of an animal model to simulate neonatal pneumothorax.

RATIONALE AND OBJECTIVES: The feasibility of developing an animal model to simulate radiographic features of neonatal pneumothorax was investigated, and supine computed radiography (CR) and film-screen radiography (FS) images were compared. METHODS: 5-Fr pigtail catheters were inserted into the right pleural space of three New Zealand white rabbits. Two CR and two FS supine images were obtained using identical technique. After serial 4-mL injections of air, one CR and one FS supine image were obtained. The images were masked and randomized, and four radiologists reviewed the images for typical signs of pneumothorax. Chest computed tomography (CT) was performed before the injection of any air and after completion of the study. RESULTS: At zero-mL air volume, radiologists did not perceive a pneumothorax. As the air volume increased, the number of signs of a pneumothorax increased; there was no significant difference between CR and FS in the number of signs of pneumothorax detected at any volume. Chest CT confirmed that there was no unexplained air leak. The volume of air injected correlated with the calculated volume from the CT. CONCLUSIONS: This induced, volume-controlled, animal model simulated the supine radiographic features of neonatal pneumothorax.

Electrocautery of the tract after needle biopsy of the liver to reduce blood loss. Experience in the canine model.

RATIONALE AND OBJECTIVES: Bleeding is the most common complication of needle biopsy of the liver. This study was designed to assess the feasibility of using unipolar electrocautery to decrease bleeding. METHODS: Under general anesthesia, the livers of eight dogs were surgically exposed. A mean of 15 biopsies was obtained with 18-gauge needles from each liver by alternating biopsies without and with electrocautery. Cautery was performed by applying radiofrequency energy at 25 watts (6 animals) or 125 watts (2 animals) to the needle as the needle was withdrawn from the liver. Blood loss was measured by applying preweighed sponges to the biopsy site, then reweighing them after use. Biopsy specimens were examined by light microscopy. RESULTS: The mean (+/- SD) blood loss was 0.44 g (+/- 1.36 g) with electrocautery and 1.47 g (+/- 2.23 g) without electrocautery (P < .01). No thermal injury was noted in the biopsy specimens when cautery was applied at 25 watts. CONCLUSIONS: The application of radiofrequency current to the biopsy needle after liver biopsy is a feasible and effective method to reduce blood loss in our canine model.

Phosphor plate computed radiography: response to variation in mAs at fixed kVp in an animal model. Potential role in neonatal imaging.

Computed digital radiography offers a number of potential advantages over conventional film-screen imaging. To determine the applicability of these to portable neonatal examinations we performed a controlled prospective study comparing conventional 200 speed film-screen with a computed radiography unit in an animal model. Images acceptable for diagnostic purposes were obtained on the digital system over an exposure range many orders of magnitude greater than was possible on the film-screen system. Digital imaging offers potential for elimination of repeat examinations due to suboptimal exposure factors, and for reduction in radiation dose to the patient. We believe that computed digital radiography should be particularly suited to portable neonatal imaging.

Comparison of computerized digital and film-screen radiography: response to variation in imaging kVp.

A controlled prospective study, in an animal model chosen to simulate portable neonatal radiography, was performed to compare the response of the Philips Computed Radiography (CR) system and conventional 200 speed film-screen (FS) to variation in imaging kVp. Acceptable images were obtained on the CR system over a very wide kVp range. In contrast the FS system produced acceptable images over a narrow kVp range. This ability suggests that the CR system should eliminate the need for repeat examinations in cases where a suboptimal kVp setting would have resulted in an unacceptable FS image. CR technology should therefore be ideally suited to portable radiography especially in situations where selection of correct exposure factors is difficult as in the neonatal nursery.

Apicoaortic valved conduits in a canine model.

Evaluating new developments in surgical methods and materials of aortic bypass prosthetics requires the use of an appropriate animal model, a well established surgical protocol and post surgical aortography. This article describes the surgical technique, post operative care and the angiographic technique used for an apicoaortic conduit placement in a canine model.