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.

Effect of ischemia and reperfusion on neutrophil accumulation in equine microvascular tissue flaps.

OBJECTIVE: To investigate neutrophil accumulation after ischemia and reperfusion (IR) in microvascular tissue flaps in horses. STUDY DESIGN: Randomized controlled experiment. SAMPLE POPULATION: A total of 8 horses between 1 and 10 years of age, 4 of each sex. METHODS: Control and experimental myocutaneous island flaps based on the superficial branch of the deep circumflex iliac vessels were dissected on each horse. Atraumatic vascular clamps were applied to the pedicle of the experimental flap for 90 minutes and then removed to allow reperfusion. Based on the assumption that rapid infiltration of neutrophils into affected tissues is a hallmark of IR injury, radiolabeled autogenous leukocytes were used to indirectly quantify neutrophil accumulation in flap tissues. Labeled leukocytes were administered through a jugular catheter 30 minutes before flap reperfusion. Biopsies were collected from each flap over a 6 hour postischemia time period; in group 1 (n = 4) from 0 to 6 hours postischemia, and in group 2 (n = 4) from 24 to 30 hours postischemia. Biopsies were examined scintigraphically and histologically for evidence of neutrophil infiltration. RESULTS: All control flaps survived and 6 of 8 experimental flaps survived. There was no significant evidence of acute neutrophil infiltration into flap tissues after reperfusion in either group. CONCLUSIONS: The results of this study suggest that equine myocutaneous flap tissues can survive a 90-minute ischemic period and reperfusion. No significant evidence of the occurrence of IR injury in flap tissues was found. CLINICAL RELEVANCE: The reasons for the previously reported failures of equine free tissue transfer remain uncertain, but they do not appear to be caused by neutrophil mediated injury associated with ischemia and reperfusion.

Microvascular free tissue transfer: results in 57 consecutive cases.

OBJECTIVE: To evaluate the outcomes and complications in a consecutive series of animals undergoing microvascular reconstructive procedures at two veterinary institutions. STUDY DESIGN: Retrospective study. ANIMALS OR SAMPLE POPULATION: A total of 44 client-owned dogs and one red-necked wallaby. METHODS: The medical records of all animals undergoing reconstructive microsurgical procedures at the Western College of Veterinary Medicine and Michigan State University were reviewed. Microvascular flap survival and related complications were described. Statistical analysis was performed to determine the significance of relationships between operative factors and outcome. RESULTS: A total of 57 microvascular procedures were performed on 55 animals. Reconstruction was required after trauma in 42 animals, after ablative cancer surgery in 11 animals and for correction of congenital tissue aplasia in I animal. Donor tissues included the superficial cervical cutaneous, medial saphenous fasciocutaneous or musculofasciocutaneous, caudal superficial epigastric cutaneous, trapezius muscle or musculocutaneous, caudal sartorius muscle, latissimus dorsi muscle or musculocutaneous, cranial abdominal myoperitoneal, carpal footpad, digital footpad, and vascularized ulnar bone flaps. A total of 53 of 57 flaps (93%) survived. There was a significant relationship between flap failure and level of assistant surgeon experience (P < .05). Latissimus dorsi flaps were significantly more likely to fail when compared with pooled data from all other flap types (P < .01). CONCLUSIONS: The success of microvascular tissue transfer in this case series compares favorably with those reported in human reconstructive microsurgery. Both the primary and assistant surgeon should be practiced in microsurgical technique. Failure of latissimus dorsi flaps was not likely caused by an inherently deficient flap design, but was more likely attributed to the location and severity of trauma at the recipient site, the difficulty in isolating suitable recipient vessels for anastomosis or the absence of a trained assistant surgeon during these procedures. Clinical Relevance-This retrospective study documents the successful application of microvascular technique in a series of clinical cases requiring tissue reconstruction.

Open-heart correction of tetralogy of Fallot in an acyanotic dog.

Tetralogy of Fallot was diagnosed in an acyanotic 11-month-old dog. Predicted pressure gradient across the pulmonic valve, as assessed by use of continuous wave Doppler echocardiography, was 94.5 mm Hg. Bidirectional shunting was identified by means of selective angiography. Open-heart correction was performed, using a transatrial approach with limited ventriculotomy and cardiopulmonary bypass. The hypertrophied infundibulum was resected, the ventricular septal defect was closed primarily, and a transannular pericardial patch graft was applied. Pressure gradients across the pulmonic valve were 52.9 and 22.8 mm Hg 2 weeks and 4 months after surgery, respectively. Advances in cardiopulmonary bypass, anesthetic management, and use of the transatrial approach may improve the success of open-heart correction of tetralogy of Fallot in dogs.

Deep hypothermic low flow cardiopulmonary bypass in small dogs.

OBJECTIVE: To evaluate the feasibility of and morbidity and mortality associated with cardiopulmonary bypass (CPB) using deep hypothermia and low flow perfusion in adult dogs weighing less than 10 kg. STUDY DESIGN: Prospective, descriptive study. ANIMALS: Two groups of three dogs underwent CPB. Group 1 dogs underwent deep hypothermia (15 to 18 degrees C), 45 minutes of low perfusion flow (20 mL/kg/min) and 1 hour of aortic cross clamp time. In group 2, ultrafiltration of perfusate before discontinuation of bypass was added to the standard treatment. Complete blood counts, serum biochemistry, urine output, ejection fraction, and cardiac output were monitored before and for 7 days after surgery. RESULTS: All dogs were successfully weaned from bypass. Four of six dogs survived, three without major complications. One dog developed and recovered from septic pleuritis. Two dogs died or were euthanatized after surgery because of respiratory or gastrointestinal complications. Minor complications included anemia, hypoproteinemia, and electrolyte disturbances. Transfusion requirements and edema formation were reduced by ultrafiltration. CONCLUSIONS: The observations in this study support the feasibility of low flow hypothermic CPB. Meticulous tissue handling, precise equipment, ultrafiltration, and aggressive postoperative potassium supplementation are recommended for smaller patients. CLINICAL RELEVANCE: Increased sensitivity to adverse sequelae of CPB may be associated with small patient size. Further evaluation is necessary before routine clinical application of low flow hypothermic CPB in this patient population.

Focal myasthenia gravis as a paraneoplastic syndrome of canine thymoma: improvement following thymectomy.

A 10-year-old, neutered male cocker spaniel-cross experienced regurgitation, dry retching, and weight loss. A large, mediastinal mass and dilatation of the esophagus were seen on thoracic radiographs. Cytological, histopathological, immunohistochemical, and serological findings were consistent with a lymphoepithelial thymoma and focal, esophageal myasthenia gravis. Surgical removal of the mass resulted in rapid resolution of the megaesophagus and a decrease in serum acetylcholine-receptor antibody concentration. The dog was clinically normal until the thymoma recurred six months postoperatively. Clinical signs, diagnostic evaluation, management, and treatment of a dog with thymoma and megaesophagus are described.

Development of a free latissimus dorsi muscle flap in cats.

Anatomic and experimental evaluation of the feline latissimus dorsi muscle was performed to assess its potential use as a free muscle flap. In the anatomic study, nonselective angiography of the subscapular artery was performed in nine heparinized feline cadavers. The muscle dimensions and vascular anatomy of the dissected latissimus dorsi muscle were recorded. In the experimental study four cats underwent heterotopic transplantation of a partial latissimus dorsi flap, and three cats underwent orthotopic transplantation of a complete latissimus dorsi flap. The mean length and width of the latissimus dorsi muscle was 19.0 and 5.4 cm, respectively. The dominant vascular pedicle was the thoracodorsal artery and vein. The average length and diameter of the thoracodorsal artery was 2.7 cm and 0.6 mm, respectively. Minor vascular pedicles were provided by branches of the intercostal arteries. Numerous choke anastomoses existed between the two pedicle systems. Viability of muscle flaps based on subjective evaluation, angiography, and histopathology, was 66% and 100% in the heterotopic and orthotopic studies, respectively. Flap failure seemed to be caused by both arterial and venous thrombosis. The latissimus dorsi muscle flap met criteria required for application in microvascular reconstruction. The vascular pattern was appropriate and consistent. Donor site morbidity was low, whereas surgical accessibility was high. The muscle satisfied the physical criteria of a free flap. Long-term anastomotic patency and flap viability was shown.

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.

Short-term response of epiphyseal plate cell populations following selective devascularization and microsurgical revascularization.

The distal femoral epiphyseal plates of 21 8-week-old New Zealand white rabbits were totally or partially (nutrient artery only) devascularized, or devascularized and then microsurgically revascularized. Sacrifice was at 24, 48 or 72 hours postoperatively. The heights of the proliferative and hypertrophic zones of the epiphyseal plates operated upon were compared with the contralateral control epiphyseal plates for both the central and the peripheral regions of the epiphyseal plate. Neither extent of devascularization nor revascularization had a significant effect on the height of the proliferative zone of chondrocytes at any of the follow-up intervals. Selective devascularization of the nutrient artery led to a significant increase in height of the central region of the hypertrophic zone of chondrocytes at 48 and 72 hours. Microsurgical revascularization did not lead to a significant change in the height of either the central or the peripheral regions of the hypertrophic zone of chondrocytes at any of the follow-up intervals. This study is another 'building block' experiment toward vascularized epiphyseal plate transplantation in humans.

The trapezius osteomusculocutaneous flap in dogs.

A pedicled osteomusculocutaneous flap, composed of the cervical part of the trapezius muscle with its overlying skin and the central spine and body of the scapula, was elevated on the prescapular branch of the superficial cervical vascular pedicle in four dogs. The flap was replaced in an orthotopic location. Bone viability was evaluated using histology, fluorescence bone labeling, and angiography. Bone from the scapular spines had a high percentage of viable osteocytes, positive fluorescence, and vessels were outlined in the angiographic study. Bone from the body of the scapula was not viable based on similar criteria.

Immunoscintigraphic detection of primary and metastatic spontaneous canine osteosarcoma with F(ab')2 fragments of osteosarcoma-associated monoclonal antibody TP-1.

Monoclonal antibody TP-1 has been shown to bind selectively to human and canine osteosarcoma cells in vitro using immunohistochemical stains. This report describes the in vivo administration of radioiodinated F(ab')2 fragments of monoclonal antibody TP-1 in dogs with primary and/or metastatic spontaneous osteosarcoma. Two dogs were injected with 131labeled F(ab')2 TP-1 and two dogs were injected with 123labeled antibody fragments. Immunoscintigraphy successfully demonstrated the radiolabeled antibody fragments in 6/6 known primary or metastatic lesions and in addition detected 4 metastatic lesions not diagnosed by conventional radiographs. Concurrent imaging of 99mTc labeled autologous erythrocytes in two dogs confirmed that the accumulation of radiolabeled antibody fragments was independent of the blood pool. There was no immunoscintigraphic evidence of localization of radioiodine to normal organs or tissues other than those expected to accumulate free iodine. The present study has demonstrated the potential of monoclonal antibody TP-1 F(ab')2 fragments for early detection of metastatic spread of spontaneous osteosarcoma.

Free microvascular transplantation of the trapezius musculocutaneous flap in dogs.

A musculocutaneous flap based on the prescapular branch of the superficial cervical artery and including the cervical part of the trapezius muscle and overlying skin was transplanted over a defect created on the medial side of the contralateral tibia in four dogs by using microvascular technique. The donor and recipient sites in three dogs were examined clinically for 21 days, after which they were examined angiographically and histologically. All dogs were free of lameness by hour 48. Seromas formed at the donor site between days 7 and 15. One vascular pedicle was traumatized at hour 40, and the dog was euthanatized. Three flaps survived with minimal necrosis. Edema of the flaps was severe from days 5 to 11. Angiograms showed complete perfusion of the flaps, and survival was confirmed histologically. Esthetic appearance and function were good in one dog at month 7.

The anatomic basis for a trapezius muscle flap in dogs.

The anatomy of the cervical part of the trapezius muscle and its dominant vascular supply, the prescapular branch of the superficial cervical artery, was studied by dissection and selective angiography of 16 canine cadavers. The prescapular branch of the superficial cervical artery supplies blood to the skin of the caudal half of the neck and the cervical part of the trapezius muscle and is a minor contributor to other muscles of the neck. In these dogs, the mean length of the vascular pedicles was 4.4 cm and the mean diameter was 1.0 mm. With this information, it is possible to design a broad musculocutaneous flap suitable for reconstructive microsurgery in dogs. The potential for successful incorporation of the scapular spine in such a flap remains uncertain.

Physiologic alteration of fibrinogen levels: influence upon patency of microvenous anastomoses.

High baseline levels of plasma fibrinogen have been correlated with increased risk for cardiovascular disease. Since fibrinogen plays a central role in both coagulation and platelet aggregation and is a primary component of thrombi, this study was designed to assess the relationship of circulating fibrinogen concentration and vein anastomotic patency. Subcutaneously injected turpentine was used to increase, and intravenous ancrod (snake venom) to decrease, plasma fibrinogen levels. Rat femoral vein anastomoses were performed, and patency was assessed after 120 min of flow. Rat tail bleeding times were obtained, and blood samples were drawn to determine fibrinogen and plasma protein concentrations, Lee-White clotting times, and activated partial thromboplastin times (APTT). Increased patency was found in the ancrod group (88%) (P < 0.05 vs. controls); turpentine-treated and control groups were not significantly different (71% and 63%, respectively). The ancrod group also showed significantly prolonged tail bleeding times and APTT. Fibrinogen levels were significantly decreased in the ancrod group (1.73 mg/ml) and elevated in the turpentine group (4.91 mg/ml) vs. controls (2.34 mg/ml; P < 0.005). These results indicate that elevated fibrinogen levels, in particular when triggered by an acute-phase response, do not appear to predispose small vessel repairs toward thrombosis. Furthermore, this study supports the use of ancrod as an anticoagulant for microvascular surgery.

Metabolic behavior of immobilized aggregates of Escherichia coli under conditions of varying mechanical stress.

Experiments were conducted on immobilized aggregates of Escherichia coli cells. Mechanical stress was applied by forcing a convective stream of nutrient medium through the aggregate. It was shown to be possible to maintain uniform exponential growth with this convective supply of nutrients. Analysis of effluent from the system allowed investigation of metabolic responses unambiguously attributable to mechanical stress. A reversible increase in catabolic activity was observed after an increase in mechanical stress. Changes in the level of catabolism were accompanied by an alteration in the total acid yield on glucose and in the spectrum of organic acids produced during glucose fermentation. The behavior observed here was likely due to an osmoregulatory response induced by the mechanically stressed bacteria to counteract changes in shape.

Hydraulic permeability of immobilized bacterial cell aggregates.

A dense aggregate of cells was retained in a reactor by a supported porous membrane. A continuous flow of nutrient medium was maintained through the cell aggregate and membrane. The hydraulic resistance of the cell aggregate was monitored throughout experiments with either growing or chemically cross-linked cells, under conditions of varying flow rates. Digital image analysis was used to characterize the sizes, separations, and orientations of several thousand individual cells in electron micrographs of chemically cross-linked cell aggregates. Two nonlinear phenomena were observed. First, the hydraulic resistance varied in direct relation to and reversibly with flow rate. Second, in constant flow-rate experiments the hydraulic resistance increased with time at a faster rate than could be attributed to cell growth. Both of these phenomena were dependent upon and could be explained by the ability of cells to move with respect to one another, under the influences of Brownian motion and of convection. Such relative motion could allow changes in net alignment of cells in the direction of flow and in the volume fraction of cells in the aggregate. This explanation is consistent with image analysis data. The observed sensitivity of hydraulic resistance to flow rate was inconsistent with a model that assumed elastic deformation of individual cells, and no evidence of cell deformation was found in electron micrographs.

Free tissue transfer of digital food pads for reconstruction of the distal limb in the dog.

Reconstructive procedures used in salvaging distal limb injuries in the dog are reviewed. Techniques employed to resurface the distal weight-bearing surface include local reconstruction using pedicle flap transposition of foot pads. The development of a previously described free microneurovascular digital foot pad flap is reviewed; the experimental results and potential for clinical use of the free flap are discussed.