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Biomedical subjects

B C Cooley

Publications and source records attributed to B C Cooley.

At least 19 recordsLinked to original sources
PubMed2001

Multiple combinations of alternatively spliced exons in rat tropomyosin-alpha gene mRNA: evidence for 20 new isoforms in adult tissues and cultured cells.

Previous studies of the tropomyosin-alpha gene using Northern blot and ribonuclease protection assay methods identified the expression of nine isoforms generated by alternative splicing of exons. Several of these isoforms were characterized as tissue-specific and/or developmentally specific. The present study used a highly sensitive RT-PCR-based strategy to assay the expression of these and many novel isoforms in a variety of adult rat tissues. All 9 isoforms were found to be expressed in all tissues evaluated. Furthermore, 20 new isoforms were identified with varying tissue specificity. Sequence analysis confirmed exon splicing patterns. This greater degree of isoform generation parallels recent findings for another tropomyosin gene, the TM-5 gene, for which the generation of new isoforms, in particular, ones using novel junctions for carboxy-terminal-coding exons, was also shown. Several of the new cDNA-based isoforms predict tropomyosin protein species that are 10 amino acids longer than previously characterized high-molecular-weight tropomyosin-alpha gene isoforms. The apparent lack of significant tissue specificity in the expression of tropomyosin isoforms suggests that many of these isoforms have more generic roles in cell function.

Alternative Splicing↗
PubMed2001

A phase II trial of intraluminal irrigation with recombinant human tissue factor pathway inhibitor to prevent thrombosis in free flap surgery.

A multicenter, multinational, blinded, randomized, parallel-group, phase II study was conducted to investigate the use of recombinant human tissue factor pathway inhibitor (rhTFPI; SC-59735) as an antithrombotic additive to the intraluminal irrigating solution during microvascular anastomosis in free flap reconstructive surgery. A total of 622 patients undergoing free flap reconstruction were randomly assigned to three groups. For each group, a different intraluminal irrigating solution was administered at completion of the microvascular arterial and venous anastomoses and before blood flow to the flap was reestablished: rhTFPI at a concentration of 0.05 or 0.15 mg/ml (low-dose or high-dose group, respectively) or heparin at a concentration of 100 U/ml (current-standard-of-practice group). There were no other differences in treatment among the groups. Patient characteristics, risk factors, and surgical techniques used were similar among all three groups. Flap failure was lower (2 percent) in the low-dose rhTFPI group than in the high-dose rhTFPI (6 percent) and heparin (5 percent) groups, but this difference was not statistically significant (p = 0.069). There were no significant differences in the rate of intraoperative revisions of vessel anastomoses (11 percent, 12 percent, and 13 percent) or postoperative thrombosis (8 percent, 8 percent, and 7 percent) among the low-dose rhTFPI, high-dose rhTFPI, and heparin groups, respectively. The rate of postoperative wound hematoma was significantly lower in the low-dose rhTFPI group (3 percent) than in the high-dose rhTFPI (8 percent) and heparin (9 percent) groups (p = 0.040). There were no differences in blood chemistry or coagulation values among the three study groups. Other than hematomas, there were no differences in the incidence or severity of adverse reactions among the three groups. It is concluded that use of rhTFPI as an intraluminal irrigant during free flap reconstruction is safe, well tolerated, and as efficacious as use of heparin for preventing thrombotic complications during and after the operation. Furthermore, the lower dose of rhTFPI (0.05 mg/ml) may reduce the occurrence of postoperative hematoma and help prevent flap failure.

Adolescent↗
PubMed2001

Characterization of a mouse model for thrombomodulin deficiency.

Mutations in the gene encoding thrombomodulin (TM), a thrombin regulator, are suspected risk factors for venous and arterial thrombotic disease. We have previously described the generation of TM(Pro/Pro) mice carrying a TM gene mutation that disrupts the TM-dependent activation of protein C. Here, it is shown that inbred C57BL/6J TM(Pro/Pro) mice exhibit a hypercoagulable state and an increased susceptibility to thrombosis and sepsis. Platelet thrombus growth after FeCl(3)-induced acute endothelial injury was accelerated in mutant mice. Vascular stasis after permanent ligation of the carotid artery precipitated thrombosis in mutant but not in normal mice. Mutant mice showed increased mortality after exposure to high doses of endotoxin and demonstrated altered cytokine production in response to low-dose endotoxin. The severity of the hypercoagulable state and chronic microvascular thrombosis caused by the TM(Pro) mutation is profoundly influenced by mouse strain-specific genetic differences between C57BL/6 and 129SvPas mice. These data demonstrate that in mice, TM is a physiologically relevant regulator of platelet- and coagulation-driven large-vessel thrombosis and modifies the response to endotoxin-induced inflammation. The phenotypic penetrance of the TM(Pro) mutation is determined by as-yet-uncharacterized genetic modifiers of thrombosis other than TM.

Animals↗
PubMed2000

The Egr-1 promoter contains information for constitutive and inducible expression in transgenic mice.

Egr-1 is an immediate early gene that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene is expressed in many cell types and is induced by a wide variety of extracellular signals. The mechanisms by which the Egr-1 gene is regulated in vivo remain poorly understood. In this study, we have generated transgenic mice with a construct containing 1200 bp of the mouse Egr-1 promoter coupled to nuclear localized LacZ. In multiple independent lines of mice, reporter gene expression was detected in subsets of endothelial cells, vascular smooth-muscle cells, cardiomyocytes, neurons, and hepatocytes. This pattern closely resembled that of the endogenous gene. After partial hepatectomy, reporter gene activity was upregulated between two- and fivefold in regenerating livers. Taken together, these findings suggest that the Egr-1 promoter contains information for appropriate spatial and temporal expression in vivo.

Animals↗
PubMed1999

Brief ex vivo perfusion with heparinized and/or citrated whole blood enhances tolerance of free muscle flaps to prolonged ischemia.

This study investigated the use of heparinized and/or citrated whole blood as a perfusate for enhancing muscle tolerance to warm ischemia. Unilateral cutaneous trunci muscle flaps were harvested from Sprague-Dawley rats and stored for 10 hr at 22-24 degrees C prior to transplantation to the groin. One group served as a non-perfused control. In three experimental groups, the flaps were hand-perfused ex vivo with 1.0 ml of heparinized, citrated, or heparinized and citrated autogenous whole blood at physiological pressures. Perfusion was administered over a 10-min period 5 hr into the ischemic period. Flaps were revascularized on the femoral vessels and then harvested 48 hr following revascularization. Tissue injury was assessed by calculation of flap weight change (indicator of tissue edema), histochemical evaluation of muscle dehydrogenase activity (nitroblue tetrazolium assay), and light microscopy. All perfused groups had significantly higher muscle dehydrogenase activity compared with non-perfused controls (P < 0.005). Perfusion with combined heparin-citrated blood was significantly more protective than perfusion with either anticoagulant alone (P < 0.025). The only statistically significant reduction in percent flap edema was seen in the combined heparin-citrate perfusion of flaps compared with nonperfused controls (P < 0.05). Histologic evaluation confirmed a reduction in tissue edema in the perfused flaps. We conclude that mid-ischemic perfusion with heparinized and/or citrated blood limits the deleterious effects of extended warm ischemia.

Analysis of Variance↗
PubMed1998

History of vein grafting.

Microsurgical vein grafting for vessel reconstruction has its roots in the vascular surgery of 100 years ago. It was not until the 1950s that vein grafts became more common for treating traumatic, atherosclerotic, and aneurysmal lesions. The success and popularization of extremity replantation quickly led to the utilization of vein grafts in select cases. Current practices use vein grafts whenever needed to bridge gaps in vessel continuity during reconstructive microsurgery.

History, 19th Century↗
PubMed1998

Free flap transplantation in mice.

A new model is presented that adapts the standard experimental groin free flap model to the mouse. The femoral vessels upon which the microvascular anastomoses are based are very small (0.2-0.4 mm diameter in the artery), making this a technically challenging exercise. A 100% patency was achieved for flap replantation in ICR (outbred) mice. Success rates for flap transplantation in Balb/C (syngeneic) mice rose from 20 to 75% with modification of the anastomoses from end-to-end to end-to-side procedures. This model offers new avenues of investigation when combined with recently developed transgenic mouse models.

Anastomosis, Surgical↗
PubMed1998

A prospective study of microvascular free-flap surgery and outcome.

Over a 6-month period, 23 members of the International Microvascular Research Group participated in a prospective survey of their microvascular free-flap practice. Data were recorded with each case for 60 variables covering patient characteristics, surgical technique, pharmacologic treatment, and postoperative outcome. A total of 493 free flaps were reported with a representative demographic distribution for age, sex, indications for surgery, risk factors, flap type, surgical technique, and pharmacologic intervention. Mixed effects logistic regression modeling was used to determine predictors of flap failure and associated complications. The overall incidence of flap failure was 4.1 percent (20 of 493). Reconstruction of an irradiated recipient site and the use of a skin-grafted muscle flap were the only statistically significant predictors of flap failure, with increased odds of failure of 4.2 (p = 0.01) and 11.1 (p = 0.03), respectively. A postoperative thrombosis requiring re-exploration surgery occurred in 9.9 percent of the flaps. The incidence of this complication was significantly higher when the flap was transferred to a chronic wound and when vein grafts were needed, with increased odds of failure of 2.9 (p = 0.02) and 2.5 (p = 0.02), respectively. There was a lower incidence of postoperative thrombosis when rectus/transverse rectus abdominis muscle (TRAM) flaps were used, where odds of failure decreased by 0.36 (p = 0.04), and when subcutaneous heparin was administered in the postoperative period, where odds decreased by 0.27 (p = 0.04). There was an overall 69-percent salvage rate for flaps identified with a postoperative thrombosis. Intraoperative thrombosis occurred in 41 cases (8.3 percent) and was observed more frequently in myocutaneous flaps or when vein grafts were needed (5.5 and 5.0 greater odds, respectively; p < 0.001) but was not associated with higher flap failure (2 of 41 cases; 4.9-percent failure rate). The incidence of a hematoma and/or hemorrhage was increased in obese patients and when vein grafts were needed [2.7 (p = 0.02) and 2.6 (p = 0.03) greater odds, respectively], whereas this complication was significantly decreased in muscle flaps (myocutaneous or skin-grafted muscle), in tobacco users, when a heparinized solution was used for general wound irrigation, and when the attending surgeon performed the arterial anastomosis (in contrast to the resident or fellow on staff) (p < 0.05 for each factor). With the multivariable analysis, many factors were found not to have a significant effect on flap outcome, including the recipient site (e.g., head/neck, breast, lower limb, etc.); indications for surgery (trauma, cancer, etc.); flap transfer in extremes of age, smokers, or diabetics; arterial anastomosis with an end-to-end versus end-to-side technique; irrigation of the vessel without or with heparin added to the irrigation solution; and a wide spectrum of antithrombotic drug therapies. These results present a current baseline for free-flap surgery to which future advances and improvements in technique and practice may be compared.

Adolescent↗
PubMed1996

Heat-induced tissue fusion for microvascular anastomosis.

Laser tissue welding was compared with a crude method of bipolar coagulator-generated heat application for achieving the same heat-induced welding effect in rat microarterial anastomoses. Rat femoral arteries were anastomosed with three triangulated stay sutures and subsequent laser welding or bipolar coagulator application between each pair of stitches. Control (non-welded) vessels received nine stitches placed circumferentially. Laser-welded vessel patency at 1 or more days postoperatively was 90% (65/72) for vessels treated with 0.1-second laser pulses, not significantly different from controls (100%; 16/16) or coagulator-welded anastomoses (88%; 14/16). Pseudoaneurysm rates were higher in the welded vessels (9% and 14% for laser- and coagulator-treated vessels, respectively) than in controls (0%). Histologic and electron microscopic evaluation revealed good healing with no apparent differences between laser- and coagulator-welded repairs. These findings suggest that laser application for microvascular tissue welding is similar to poorly controlled welding with a bipolar coagulator.

Anastomosis, Surgical↗
PubMed1996

Comparison of arterial and venous patency in a rat model of subendothelium-stimulated thrombosis.

The effectiveness of anticoagulants and platelet aggregation inhibitors was compared using comparable rat models of arterial and venous thrombosis. A mechanical endothelium-denuding injury was created on the lumenal surface of donor Lewis rat carotid arteries. These were cut into 4-5 mm lengths and grafted into femoral veins and arteries of recipient syngeneic rats using microvascular anastomotic techniques. Recipients received either systemic heparin, or aspirin with dipyridamole, or saline (control). In the arteries, the 1-day patency rate was 94% in the heparin-treated rats, but only 50% in the aspirin/dipyridamole group and 44% in the control group. The venous patency rate was 56% in the heparin group, 31% in the aspirin/dipyridamole group, and 0% in the control group. This unique model for comparing thrombosis in arteries and veins shows that anticoagulation is more effective than inhibition of platelet aggregation in the rat arterial system, with less of a differential effect in the venous system.

Animals↗
PubMed1996

Arterial thrombosis enhanced by an acute phase response.

Induction of an acute phase response causes changes in the levels of specific plasma proteins. Fibrinogen is elevated to approximately twice its normal concentration by trauma, inflammation, or infection. In this study, an acute phase response was induced by subcutaneous turpentine injection in rats. Twenty-four hours later, an arterial model of thrombosis was created bilaterally in the femoral arteries. Patency at 1 and 7 days postoperatively was 24% (9/38) in comparison to 56% (20/36) in control (saline-injected) rats undergoing the same thrombosis model. Fibrinogen levels were elevated in the turpentine group to 5.3 +/- 0.8 mg/ml at the time of the surgery and 7.2 +/- 0.3 mg/ml 24 hours after surgery. In contrast, the control group had plasma fibrinogen levels of 2.5 +/- 0.2 mg/ml at the time of surgery and 5.4 +/- 0.5 mg/ml 24 hours postoperatively. These findings suggest that when an acute phase reaction has been induced several hours prior to a microvascular procedure, there may be an increased risk for development of arterial thrombosis.

Acute-Phase Reaction↗
PubMed1996

Prevention of microvascular thrombosis by topical application of recombinant tissue factor pathway inhibitor.

Tissue factor pathway inhibitor is a naturally occurring protein inhibitor of factor X and the tissue factor-factor VII complex of the extrinsic pathway of coagulation. The potential of tissue factor pathway inhibitor as a topical antithrombotic agent was evaluated in a rabbit model of thrombosis that combined intimal injury, anastomosis, and a twisted pedicle. In 207 rabbit ears, a near-complete amputation was performed, preserving the central ear artery and vein. The central ear artery was transected, the intima was removed mechanically over a 1-cm length, the artery was anastomosed, and the ear was twisted 360 degrees, wrapping the intact vein around the artery. Before recirculation, the lumen was irrigated on a blinded, randomized basis with either hirudin (100 or 500 units/ml), heparin (50 or 100 units/ml), tissue factor pathway inhibitor (10, 40, 125, or 250 microgram/ml), heparin and tissue factor pathway inhibitor together, or vehicle (control). Upon arterial reflow, the ears were observed for 7 days. Patency rates after 7 days were as follows: hirudin, 30 and 55 percent; heparin, 43 and 50 percent; tissue factor pathway inhibitor, 75 and 90 percent; heparin and tissue factor pathway inhibitor, 75 percent; and vehicle, 6 percent. The higher concentrations of tissue factor pathway inhibitor led to significantly higher patency rates than heparin, hirudin, or control solutions. Electron microscopic evaluation of specimens irrigated with gold- labeled tissue factor pathway inhibitor revealed the inhibitor bound to the injured intimal surface for at least 3 days postoperatively. Coagulation studies showed no change in the clotting profile upon intravascular infusion with tissue factor pathway inhibitor even at the highest dose used topically. We conclude that tissue factor pathway inhibitor is a more effective topical antithrombotic agent than either heparin or hirudin.

Administration, Topical↗
PubMed1996

Repair of calvarial defects with flap tissue: role of bone morphogenetic proteins and competent responding tissues.

Bone morphogenetic proteins 2 through 8 have the ability to induce the in vivo transformation of extraskeletal mesenchymal tissue into bone. The aims of this investigation were to determine the optimal responding tissue and the specificity of the inductive effect of bone morphogenetic protein 3. The optimal responding tissue was found to be skeletal muscle. The specificity of this response to bone morphogenetic protein 3 was compared with that of recombinant human basic fibroblast growth factor, recombinant platelet-derived growth factor, and recombinant insulin-like growth factor. Bone morphogenetic protein 3 was the only factor that induced de novo bone formation. This ability to transform muscle into bone was tested in 7 x 7 mm irradiated skull defects in the rat. After 1500 rads of exposure, these defects showed no significant signs of healing by 8 months. When these defects were treated with the microvascular transfer of a nonirradiated muscle flap, they had 8 percent healing at 4 months and 37 percent healing by 8 months. Defects treated with 30 micrograms bone morphogenetic protein 3 (without the muscle flap) achieved 50 percent healing by 4 months and 64 percent healing by 8 months. When the defects were treated with both the muscle flap and bone morphogenetic protein 3, there was 96 percent healing by 4 months and 100 percent healing by 8 months (p < 0.015, compared with bone morphogenetic protein 3 alone at both time points). At 8 months, the transplanted muscle was entirely transformed into bone and healed the skull defect with newly generated bone indistinguishable from the surrounding calvarial tissue. These findings suggest a potential clinical utility of bone morphogenetic protein 3-induced bone formation in skeletal reconstructions. Furthermore, they also show that there is a collaborative requirement for both the osteoinductive factor bone morphogenetic protein 3 and the presence of competent responsive cells in the well-perfused muscle.

Animals↗
PubMed1995

Intravascular heparin protects muscle flaps from ischemia/reperfusion injury.

Heparin has been found to decrease ischemia/reperfusion injury in skeletal muscle and other tissue/organ systems. The timing of heparin administration to the muscle vasculature has not been explored. We investigated the use of heparinized blood as a washout solution during ischemia to reduce ischemia/reperfusion injury. A rat cutaneous maximus muscle free flap was subjected to a 10-hr period of room temperature ischemia, then was heterotopically transplanted to the groin via microsurgical revascularization to the femoral vessels. In three experimental groups, flaps were subjected to brief ex vivo perfusion with autologous heparinized blood, at 2, 5, or 8 hr into the 10-hr ischemic interval. In the two other groups, the flaps were not perfused, and the animals were systemically heparinized either before ischemia or before transplantation, respectively. A control group underwent no flap perfusion or systemic heparinization. After transplantation, flaps were given a 48-hr period of in vivo reperfusion, then were harvested for evaluation. Flaps undergoing ex vivo perfusion or preischemic heparinization had no significant differences in weight gain (edema) compared with flaps receiving posttransplant heparinization or no heparinization (controls). The dehydrogenase staining of muscle biopsies was significantly faster (indicative of viable tissue) for perfused flaps and the flaps for which the animals received preischemic heparinization, when compared with flaps for which the animals received posttransplant heparinization or no heparinization. From these results, we conclude that heparin offers protection from ischemia/reperfusion injury when it can be introduced into the vascular network either prior to or during the ischemia period. These findings suggest the possibility of using heparinized washout solutions to enhance survival in amputated extremities.

Anastomosis, Surgical↗
PubMed1995

Topical tissue factor pathway inhibitor improves free-flap survival in a model simulating free-flap errors.

Free flap failure is frequently due to tension, twisting, kinking, or compression of the vascular pedicle after the anastomosis is completed. A rabbit model simulating these errors was used to evaluate the capacity of topically-applied tissue factor pathway inhibitor (TFPI) to prevent microvascular thrombosis. The rabbit ear was isolated on the central artery and vein. The artery was transected, shortened, repaired, and twisted 360 degrees around the vein. Immediately following the anastomosis. TFPI in concentrations of 1, 4, 10, or 40 micrograms/ml was irrigated across the lumen. Topically-applied control buffer and heparin (50 U/ml) were compared to TFPI. Treatment with control buffer resulted in a 20 percent survival rate. Topically-applied heparin improved the survival rate to 60 percent (p < 0.05). In contrast, TFPI in concentrations of 4, 10, and 40 micrograms/ml yielded survival rates of 89, 100, and 97 percent, respectively. This was significantly greater than the heparin-treated ears (p < 0.05). TFPI in a concentration of 40 micrograms/ml was effective in preventing arterial thrombosis when applied for as little as 30 sec; 4 micrograms/ml was effective in preventing thrombosis when applied for 10 min. These results support the use of TFPI as a topical irrigation solution to help prevent microvascular arterial thrombosis in free-flap surgery.

Administration, Topical↗