Lyophilized allografts without pre-treatment with glutaraldehyde are more suitable than cryopreserved allografts for pulmonary artery reconstruction.

Various methods are available for preservation of vascular grafts for pulmonary artery (PA) replacement. Lyophilization and cryopreservation reduce antigenicity and prevent thrombosis and calcification in vascular grafts, so both methods can be used to obtain vascular bioprostheses. We evaluated the hemodynamic, gasometric, imaging, and macroscopic and microscopic findings produced by PA reconstruction with lyophilized (LyoPA) grafts and cryopreserved (CryoPA) grafts in dogs. Eighteen healthy crossbred adult dogs of both sexes weighing between 18 and 20 kg were used and divided into three groups of six: group I, PA section and reanastomosis; group II, PA resection and reconstruction with LyoPA allograft; group III, PA resection and reconstruction with CryoPA allograft. Dogs were evaluated 4 weeks after surgery, and the status of the graft and vascular anastomosis were examined macroscopically and microscopically. No clinical, radiologic, or blood-gas abnormalities were observed during the study. The mean pulmonary artery pressure (MPAP) in group III increased significantly at the end of the study compared with baseline (P=0.02) and final [P=0.007, two-way repeat-measures analysis of variance (RM ANOVA)] values. Pulmonary vascular resistance of groups II and III increased immediately after reperfusion and also at the end of the study compared to baseline. The increase shown by group III vs group I was significant only if compared with after surgery and study end (P=0.016 and P=0.005, respectively, two-way RM ANOVA). Microscopically, permeability was reduced by ≤75% in group III. In conclusion, substitution of PAs with LyoPA grafts is technically feasible and clinically promising.

Pulmonary perfusion during lung transplant rejection and experimental pneumonia.

Six left lung allotransplants were performed in healthy mongrel dogs. Immunosuppression was established with cyclosporine (15 mg/kg/day p.o.) from the day of transplantation for 30 days. Another group of animals (n = 3) was used to produce acute experimental pneumonia by instilling 4-6 ml of a 10(8) CFU suspension of Pseudomonas aeruginosa into the right lower lobe. Dynamic perfusory lung scintigraphy (DPLS) was performed before transplant/pneumonia (control), during acute rejection/pneumonia as detected radiologically, and after treatment with methylprednisolone (1 g/day for 3 days i.v.) (transplant group) or antibiotics (pneumonia group). Seroalbumin macroaggregates (5-8 McI) marked with 99-mTc were injected into the cephalic vein and the percentage of perfusion to each lung was determined. Eight acute rejection episodes were detected. DPLS showed similar perfusion to each lung, whereas during acute rejection perfusion was significantly reduced by almost 30%. Perfusion was reestablished to control levels after treatment with methylprednisolone. Reduction in perfusion correlated with radiological rejection grading. No reduction in left lung perfusion was detected in pneumonia animals. In conclusion, acute rejection reduces perfusion to the transplanted lung as measured by DPLS. Treatment restores normal perfusion.

Urinary thromboxane B2 as an indicator of acute rejection in lung allotransplantation.

The behavior of urinary thromboxane B2 (TXB2) during acute rejection of lung allotransplants was evaluated. Unmatched mongrel dogs were submitted to a left lung orthotopic allotransplantation (groups I and II), or a sham operation (group III). All animals had an initial significant elevation of TXB2 excretion due to surgical trauma; however, in sham-operated animals (group III) this elevation returned to basal levels after 3 days. All transplanted animals (groups I and II) had persistent TXB2 elevation with 2 important peaks on postop days 5 and 9. The elevated TXB2 excretion persisted in spite of immunosuppressive treatment with azathioprine and prednisone (group II). Rejection was followed by means of an objective grading system applied to chest roentgenograms taken on all animals. It was found that TXB2 levels correlated directly with the grade of radiographic changes seen, thus indicating degree of rejection. TXB2 can be useful as a noninvasive indicator for surveillance of lung allograft rejection.

Effect of perfusion and preservation on IL1-beta, IL-6, and IL-10 production in murine lung.

We evaluated the production of the interleukins (ILs) IL-1beta, IL-6, and IL-10 in both the vasculature and pulmonary tissue before and after 24 h of lung preservation. The cardiopulmonary blocs of 21 Balb-c mice were divided into three study groups (7 mice/group) and were flushed through the pulmonary artery with Krebs-Henseleit buffer (K-Hb) at 4 degrees C at a rate of 0.2 mL/min as follows: Group 1, lung washout: lungs were flushed until pulmonary effluent was clear. Group 2, perfusion: After lungs were flushed until pulmonary effluent was clear, lungs were perfused during 30 min. Group 3, preservation: Lungs were flushed until pulmonary effluent was clear, and the cardiopulmonary bloc was preserved immersed into (K-Hb) at 4 degrees C. After 24 h of preservation, lungs were reperfused during 30 min. In all study groups the caudal lobe from the left lung was taken for microscopical study; all other lobes were homogenized with (K-Hb) and the supernatant was obtained. IL-1beta, IL-6, and IL-10 production in lung effluents (washout, perfusion, and reperfusion) and in lung tissue were measured by enzyme-linked immunosorbent assay (ELISA). In the lung effluent, there was no statistical difference between IL-1beta and IL-6 concentrations. In all study groups, IL-10 production was significantly higher than IL-1beta and IL-6 levels. IL-10 level was lowest in the 24-h preservation group when it was compared to the other groups. In group 1, there was a negative correlation (r = -.599, p < .05) between IL-1beta and IL-10. In pulmonary tissue, IL-1beta was higher in group 2 when compared to groups 1 (p = .001) and 3 (p = .002), and it was significantly lower in group 3. IL-10 was lower in group 1 when compared to groups 2 (p = .001) and 3 (p = .004). In groups 1 and 2, IL-1beta was significantly higher than IL-6 and IL-10. In group 3, IL-10 was higher than IL-1beta (p = .0001) and IL-6 (p = .0001). Correlation of effluent/tissue index with histological findings showed a negative correlation between IL-10 effluent/tissue relation and inflammation (r = -.68, p < .01). In conclusion, the main cytokine found in lung effluents was IL-10, followed by IL-6 and IL-1beta. On the other hand, cytokine concentration in lung tissue homogenates was mainly due to the presence of IL-1beta. However, this cytokine shows a significant reduction in lung tissue after prolonged preservation.

Pulmonary arterial contribution to airway blood flow after lung transplantation in dogs.

Despite the improved success of lung transplantation, ischemia of the donor bronchus continues to be the most important factor influencing airway healing. Recent studies have shown that at the level of the mainstem bronchi the pulmonary contribution to the airway blood flow may be equivalent to or greater than the systemic contribution and could therefore assist early healing of the newly anastomosed bronchus and, in addition, might facilitate the improved healing associated with omentopexy. The aim of this study was to measure the pulmonary contribution to airway blood flow in dogs after allotransplantation of the left lung and to determine whether omentopexy might improve the healing process. Using the radioactive microsphere technique, we measured the pulmonary contribution to airway blood flow in 25 dogs 1 week after allotransplantation of the left lung. Half the dogs had an omental wrap around the anastomotic site. Results showed that pulmonary blood flow increased progressively from lower trachea to distal mainstem bronchus and supplied the left mainstem bronchus above as well as below the anastomotic site. Omentopexy did not increase flow or enhance healing.

Plasma thromboxane B2 concentrations and pulmonary vascular resistance during lung reperfusion.

The purpose of this study was to measure the behavior of plasmatic thromboxane B2 (pITxB2) after reperfusion of a glucose-insulin-potassium preserved lung. Seven adult mongrel dogs underwent a left lung allotransplantation. Hemodynamic changes including pulmonary artery pressure and cardiac output were measured. Pulmonary artery vascular resistance, systemic resistance, arterio-venous oxygen difference, and shunt were calculated. Immunoreactive arterial and venous plasma thromboxane B2 concentrations were measured at 0 (basal), 60, 120, and 180 min after reperfusion. Hemodynamic measurements were made after 5 min of occlusion of the right pulmonary artery and ventilation with 100% oxygen. Prepreservation, pre-reperfusion, and posttransplant lung weights were obtained. All animals survived the procedure. Ischemic time was 14.72 (+/-0.31) h. Cardiac output, systemic arterial pressure, and arterio-venous oxygen difference decreased while systemic vascular resistance, pulmonary vascular resistance, and shunt increased during the study. Mean pulmonary artery pressure correlated with pulmonary vascular resistance (p < .01). Oxygen tension diminished significantly at 180 min after reperfusion. Mean basal pulmonary arterial TxB2 was 3589 (+/-424) pg/ml; mean plasma pulmonary venous TxB2 was 6578 (+/-1571) pg/ml. Pulmonary arterial to venous TxB2 ratio (a/vTxB2) increased from 0.70 at basal measurement to 0.83 at 60 min, and 0.99 at 120 and 180 min after reperfusion (p < .05). Pulmonary arterial TxB2 had a positive correlation with mean pulmonary artery pressure (p < .05); also, a/v pITxB2 correlated with pulmonary vascular resistance (r = .616, p < .01). Mean post-reperfusion lung weight increase was 74.88% (45.37 g). In conclusion, pITxB2 a/v ratio ratio increases after reperfusion of a 14-h preserved lung; pulmonary vascular resistance significantly increases after 180 min of reperfusion and correlates with the increase in pITxB2 a/v ratio.

Effect of prostaglandin E2 on the tracheobronchial distribution of lung preservation perfusate.

Complete lung preservation requires the perfusate to reach the cell it intends to protect; this is directly related to the distribution of the preserving solution throughout the lung vasculature. Several prostanoids are clinically used to enhance lung preservation. We evaluated the effect of prostaglandin E2 (PGE2) on the distribution of lung perfusate throughout tracheobronchial tissue. Fourteen pulmonary blocks were procured from an equal number of dogs and divided according to whether or not they had previously received a PGE2 infusion. All lung blocks were perfused with a glucose-insulin-potassium solution, and distribution within the lung parenchyma and tracheobronchial tissue was measured using the flow reference technique and gadolinium-153-labeled microspheres. Once perfusion had taken place, samples of lung parenchyma, tracheobronchial tissue, and flow reference were measured for radioactivity, and flow was calculated per 100 g tissue. Animals receiving PGE2 had an expected 38% decrease in systemic arterial pressure; the duration of infusion of lung perfusate during procurement was shorter in those animals receiving PGE2 (5.75 +/- 0.3 min, vs. no PGE2 8.9 +/- 1.2 min; p < .05). Perfusate flow of bronchial mucosa and cartilage increased by two to three times with the infusion of PGE2 (p < .01). Perfusate flow to lobar bronchus or lung parenchyma was similar in both groups. Flow within the lung parenchyma did not differ statistically when compared to its lobar distribution. In conclusion, PGE2-treated animals had a two- to threefold increase in perfusate flow to mainstem bronchi (including mucosa); these findings to some extent support the rationale for utilizing prostanoids in order to enhance lung preservation in clinical lung transplantation.

Suture-line reinforcement with glutaraldehyde-preserved bovine pericardium for nonanatomic resection of lung tissue.

In this study we assessed the usefulness of glutaraldehyde-preserved bovine pericardium (GPBP), preparated in our laboratory, in nonanatomic resection of lung tissue in dogs. A 30% resection of the right cranial lobe of the lung was performed in 18 mongrel dogs. The suture line was reinforced with GPBP strips. For group I (n = 6), the GPBP strips were fixed on the lung with nonabsorbable suture by thoracotomy. In Group II (n = 6), the resection and fixation of the GPBP strips were performed with an endoscopic linear stapler by thoracotomy. In Group III (n = 6), the resection and fixation of the GPBP strips were performed with a linear stapler by thoracoscopy. The animals were evaluated each day during the first week after surgery and every other day during the study time. At the end of the study all animals were euthanized with an overdose of pentobarbital. Macroscopic and microscopic examinations of the bioprosthesis and lung were evaluated. All animals survived the surgical procedure and study time (8 weeks). In the three groups, macroscopic examination of the bioprosthesis showed good adaptation to the lung tissue. Microscopically, all groups of animals presented good healing, with the presence of fibrotic tissue layer on the GPBP and its periphery as well as in the lung. However, in group I we observed the presence of giant cells in the suture line. GPBP proved to be a useful material for reinforcement of the nonanatomic resection suture line of lung tissue in dogs.

Thoracoabdominal wall repair with glutaraldehyde-preserved bovine pericardium.

Glutaraldehyde-preserved bovine pericardium (GPBP) is evaluated as a bioprosthesis for the reconstruction of surgical defects in the thoracoabdominal wall. The mechanical properties of bovine pericardium preserved at different concentrations of glutaraldehyde were studied. Samples preserved in 0.5% glutaraldehyde showed a significantly higher tensile strength (11.7 +/- 0.8 N/mm2) than samples preserved in 2.5, 5, or 10% (similar to pericardium preserved in normal saline). The percentage of elongation was significantly lower than samples preserved in 1, 2.5, and 5% glutaraldehyde. GPBP at 0.5% was used to repair experimentally induced defects of the abdominal wall (n = 9), chest wall (n = 6), diaphragm (n = 6), and sternum (n = 7). All animals presented adequate tolerance to the material used and no case of infection or rejection of the material was seen in any of the animals. Finally, 0.5% GPBP was used clinically in a series of 40 patients: postincisional abdominal hernia (n = 30), inguinal hernia (n = 8), diaphragmatic hernia (n = 1), and congenital pelvic defect with prolapse of abdominal organs (n = 1). Surgical use showed that GPBP was a very manageable material and long-term results were good in 37 patients with a mean follow up of 18 months (range 5-35 months). Six patients presented seroma formation (all abdominal hernia patients), three of which eventually developed infection and had the GPBP patch removed at 3, 5, and 7 months postoperatively. The rest of the patients presented good scar formation with adequate resistance at the area of implantation. GPBP is a biological material with sufficient resistance to be used surgically in the repair of thoracoabdominal defects. Ideal concentration of glutaraldehyde to be used in the preparation-preservation of the material is 0.5% since higher concentration negatively affect its tensile rupture strength and elongation.

[Repair of thoracoabdominal wall defects in dogs using a bovine pericardium bioprosthesis]. / Reparación de defectos de pared tóracoabdominal de perros con bioprótesis de pericardio bovino.

OBJECTIVES: To evaluate the in-vitro resistance of a pericardial tissue bioprothesis as well as its use for the reconstruction of surgical defects in the thoracoabdominal wall. MATERIALS AND METHODS: The prosthesis were preserved for 15 days in different concentrations of glutaraldehyde (0.5% to 10%). Normal saline and Hanks solution were used as controls. Tensile strength and percent elongation were measured using an Instrom 1011 automatic equipment; polypropilene (Marlex) and polyester (Mersilene) mesh samples were included. Also, the 0.5% glutaraldehyde-preserved pericardium was used to repair defects experimentally produced in the abdominal wall (n = 12), chest wall (n = 6), diaphragm (n = 6), and sternum (n = 7) of mongrel dogs. RESULTS: The samples preserved in 0.5% glutaraldehyde showed a significantly higher tensile strength (11.7 N/mm2, SEM = 0.8) than samples preserved in other concentrations as well as in the mesh samples. Percent elongation in 0.5% glutaraldehyde was 56% (SEM = 5.8) which was similar to the meshes' elongation but significantly lower than that seen for the 1%, 2.5% and 5% glutaraldehyde-preserved samples. The dogs showed good tolerance to the 0.5% preparation and the histopathology showed formation of a fibroblast layer with collagen deposits and development of scar tissue. There was no case of infection or rejection in the animals, and all the microbiological cultures of the preparations were negative. CONCLUSION: Pericardial bioprothesis is a resistant material which can be used surgically in the repair of thoracoabdominal defects.

[Effect of the hyaluronic acid on tracheal healing. A canine experimental mode]. / Efecto del ácido hialurónico sobre la cicatrización traqueal en un modelo experimental canino.

Several drugs have been used to modulate of the tracheal healing process in order to prevent tracheal stenosis. Hyaluronic acid (HA) is a modulator of the fibrogenesis. In this work we evaluate the effect in order the application of hyaluronic acid has on tracheal healing, after cervical tracheoplasty in dogs. A cervical tracheal resection and tracheoplasty was performed in 12 dogs and they were treated following surgery as follows: Group I (n = 6) Topical application of normal saline solution (0.9%) on the anastomosis site. Group II Topical application of hyaluronic acid on the trachea anastomosed. The animals were evaluated clinical, radiological and tracheoscopically during 4 weeks and were submitted to euthanasia. Macroscopic and microscopic examinations of the tracheal anastomotic healing were evaluated. Biochemical collagen quantification by the Woessner method was performed to evaluate the collagen development at the anastomotic site. All the animals survived the surgical procedure and the study time. No animal presented differences in clinical evaluation. Radiological and endoscopical findings both two showed more development of the tracheal stenosis in-group than in group II. The tracheoscopy and macroscopic studies showed major inflammation and development of fibrotic tissue with a firm consistency in the healing of the group I than in group II. Microscopic examination in group I showed severe fibrosis and inflammatory reaction. The group II presented deposits of a thin and organized collagen fibers and minimal inflammatory reaction. Biochemical collagen concentration was larger in-group I, however significantly. We conclude that the hyaluronic acid applied after cervical tracheoplasty in dogs reduces postsurgical tracheal stenosis and inflammation, as well as improve the quality of the tracheal healing.

Lyophilized allografts without pre-treatment with glutaraldehyde are more suitable than cryopreserved allografts for pulmonary artery reconstruction

Various methods are available for preservation of vascular grafts for pulmonary artery (PA) replacement. Lyophilization and cryopreservation reduce antigenicity and prevent thrombosis and calcification in vascular grafts, so both methods can be used to obtain vascular bioprostheses. We evaluated the hemodynamic, gasometric, imaging, and macroscopic and microscopic findings produced by PA reconstruction with lyophilized (LyoPA) grafts and cryopreserved (CryoPA) grafts in dogs. Eighteen healthy crossbred adult dogs of both sexes weighing between 18 and 20 kg were used and divided into three groups of six: group I, PA section and reanastomosis; group II, PA resection and reconstruction with LyoPA allograft; group III, PA resection and reconstruction with CryoPA allograft. Dogs were evaluated 4 weeks after surgery, and the status of the graft and vascular anastomosis were examined macroscopically and microscopically. No clinical, radiologic, or blood-gas abnormalities were observed during the study. The mean pulmonary artery pressure (MPAP) in group III increased significantly at the end of the study compared with baseline (P=0.02) and final [P=0.007, two-way repeat-measures analysis of variance (RM ANOVA)] values. Pulmonary vascular resistance of groups II and III increased immediately after reperfusion and also at the end of the study compared to baseline. The increase shown by group III vs group I was significant only if compared with after surgery and study end (P=0.016 and P=0.005, respectively, two-way RM ANOVA). Microscopically, permeability was reduced by ≤75% in group III. In conclusion, substitution of PAs with LyoPA grafts is technically feasible and clinically promising.

Tracheal cryopreservation: caspase-3 immunoreactivity in tracheal epithelium and in mixed glands.

Cryopreservation has an immunomodulating effect on tracheal tissue as a result of class II antigen depletion due to epithelium exfoliation. However, not all epithelium is detached. We evaluated the role of apoptosis in the remaining epithelium of 30 cryopreserved tracheal grafts. Caspase-3 immunoreactivity of tracheal epithelium was studied in canine tracheal segments cryopreserved with F12K medium, with or without subsequent storage in liquid nitrogen at -196 degrees C for 15 days. Loss of structural integrity of tracheal mixed glands was observed in all cryopreserved tracheal segments. Caspase-3 immunoreactivity in tracheal mucosa and in mixed glands was significantly decreased, in contrast to the control group and to cryopreserved tracheal segments in which it remained high, due to the effect of storage in liquid nitrogen (P < 0.05, ANOVA and Tukey test). We conclude that apoptosis can be triggered in epithelial cells during tracheal graft harvesting even prior to cryopreservation, and although the epithelial caspase-3 immunoreactivity is reduced in tracheal cryopreservation, this could be explained by increased cell death. Apoptosis cannot be stopped during tracheal cryopreservation.

Tracheal cryopreservation: caspase-3 immunoreactivity in tracheal epithelium and in mixed glands

Cryopreservation has an immunomodulating effect on tracheal tissue as a result of class II antigen depletion due to epithelium exfoliation. However, not all epithelium is detached. We evaluated the role of apoptosis in the remaining epithelium of 30 cryopreserved tracheal grafts. Caspase-3 immunoreactivity of tracheal epithelium was studied in canine tracheal segments cryopreserved with F12K medium, with or without subsequent storage in liquid nitrogen at -196°C for 15 days. Loss of structural integrity of tracheal mixed glands was observed in all cryopreserved tracheal segments. Caspase-3 immunoreactivity in tracheal mucosa and in mixed glands was significantly decreased, in contrast to the control group and to cryopreserved tracheal segments in which it remained high, due to the effect of storage in liquid nitrogen (P < 0.05, ANOVA and Tukey test). We conclude that apoptosis can be triggered in epithelial cells during tracheal graft harvesting even prior to cryopreservation, and although the epithelial caspase-3 immunoreactivity is reduced in tracheal cryopreservation, this could be explained by increased cell death. Apoptosis cannot be stopped during tracheal cryopreservation.