Retrograde Flush Research Articles

Retrograde Flush Following Warm Ischemia in the Non-Heart-Beating Donor Results in Superior Graft Performance at Reperfusion

The use of non-heart-beating donors (NHBD) has been propagated as an alternative to overcome the scarcity of pulmonary grafts. The presence of postmortem thrombi, however, is a concern for the development of primary graft dysfunction. In this isolated lung reperfusion study, we looked at the need and the best route of preharvest pulmonary flush. Domestic pigs were sacrificed by ventricular fibrillation and divided in 3 groups (n = 6 per group). After 1 h of in situ warm ischemia, lungs in group I were retrieved unflushed (NF). In group II, lungs were explanted after an anterograde flush (AF) through the pulmonary artery. Finally, in group III, lungs were explanted after a retrograde flush (RF) via the left atrium. After 3 h of cold storage, the left lung was assessed for 60 min in our ex vivo reperfusion model. Wet-to-dry weight ratio (W/D) was calculated after reperfusion. Pulmonary vascular resistance (dynes x sec x cm(-5)) was 1145 +/- 56 (RF) versus 1560 +/- 123 (AF) and 1435 +/- 95 (NF) at 60 min of reperfusion (P < 0.05). Oxygenation and compliance were higher and plateau airway pressure was lower in RF versus AF and NF, although the difference did not reach statistical significance. No differences in W/D were observed between groups after reperfusion. Histological examination revealed fewer microthrombi in the left lung in RF compared with AF and NF. RF of lungs from NHBD improves graft function by elimination of microthrombi from the pulmonary vasculature, resulting in lower pulmonary vascular resistance upon reperfusion.

Donor Fat Embolism and Primary Graft Dysfunction After Lung Transplantation

Primary lung graft dysfunction is one of the major causes of perioperative morbidity and mortality in lung transplantation. Primary lung graft dysfunction is a clinical syndrome occurring in the immediate postoperative period after lung transplantation and is characterized by severe hypoxemia, pulmonary edema, and pulmonary infiltrates on chest x-ray film, requiring that the patient remain intubated and thus favoring pulmonary infection, sepsis, and subsequent multiple organ failure in the transplanted patient. It has recently been shown that unexpected pulmonary embolism is relatively common in the donor and is associated with primary lung graft dysfunction. However, we believe that only one case of primary lung graft dysfunction due to pulmonary fat embolism has been documented histologically in patients undergoing lung transplantation. The objective of this study is to report our experience with a case of primary lung graft dysfunction due to fat embolism in the donor lung detected in the morphologic study.

Retrograde flush following topical cooling is superior to preserve the non-heart-beating donor lung☆☆☆

The use of non-heart-beating donors (NHBD) has been propagated as an alternative to overcome the scarcity of pulmonary grafts. Formation of microthrombi after circulatory arrest, however, is a major concern for the development of reperfusion injury. We looked at the effect and the best route of pulmonary flush following topical cooling in NHBD. Non-heparinized pigs were sacrificed by ventricular fibrillation and divided into three groups (n=6 per group). After 1h of in situ warm ischaemia and 2.5h of topical cooling, lungs in group I were retrieved unflushed (NF). In group II, lungs were explanted following an anterograde flush (AF) through the pulmonary artery with 50 ml/kg Perfadex (6 degrees C). Finally, in group III, lungs were retrieved after an identical but retrograde flush (RF) via the left atrium. Flush effluent was sampled at intervals to measure haemoglobin concentration. Performance of the left lung was assessed during 60 min in our ex vivo reperfusion model. Wet-to-dry weight ratio (W/D) of both lungs was calculated as an index of pulmonary oedema. IL-1beta and TNF-alpha protein levels in bronchial lavage fluid from both lungs were compared between groups. Haemoglobin concentration (g/dl) was higher in the first effluent in RF versus AF (3.4+/-1.1 vs 0.6+/-0.1; p<0.05). Pulmonary vascular resistance (dynes x s x cm(-5)) was 975+/-85 RF versus 1567+/-98 AF and 1576+/-88 NF at 60 min of reperfusion (p<0.001). Oxygenation (mmHg) and compliance (ml/cmH(2)O) were higher (491+/-44 vs 472+/-61 and 430+/-33 NS, 22+/-3 vs 19+/-3 and 14+/-1 NS, respectively) and plateau airway pressure (cmH(2)O) was lower (11+/-1 vs 13+/-1 and 13+/-1 NS) after RF versus AF and NF, respectively. No differences in cytokine levels or in W/D ratios were observed between groups after reperfusion. Histology demonstrated microthrombi more often present after AF and NF compared to RF. Retrograde flush of the lung following topical cooling in the NHBD results in a better washout of residual blood and microthrombi and subsequent reduced pulmonary vascular resistance upon reperfusion.

308: Elevated IL-8 levels in retrograde flush effluent is associated with unexpected donor pulmonary embolism: A novel predictor for primary graft dysfunction?

Purpose: Unexpected donor pulmonary embolism (PE) is relatively common in lung transplantation (LTx). We have previously suggested that unexpected PE is associated with primary graft dysfunction (PGD) and potentially can be predicted by donor history (Oto JTCVS 2006). Diagnosing unexpected PE and preventing PGD is mandatory for successful early outcomes after LTx. We investigated inflammatory cytokines in donor lung retrograde flush effluent and correlated this with unexpected PE and recipient post-transplant oxygenation.

Lung Preservation: Pulmonary Flush Route Affects Bronchial Mucosal Temperature and Expression of IFN-γ and Gro in Regional Lymph Nodes

Optimal lung preservation via flush of the pulmonary vasculature minimizes early graft failure post-lung transplantation. We hypothesized that the route of pulmonary flush has differential effects on thermal gradients in the lung and expression of inflammatory mediators. Swine underwent antegrade flush (AG) via pulmonary artery; AG/RG: antegrade + retrograde flush via pulmonary veins or AG/BA: antegrade + bronchial artery flush via bronchial artery. Temperatures were recorded in bronchial mucosa and peribronchial lymph nodes. RT-PCR was utilized to detect cytokine gene expression in the nodes. AG/BA flush resulted in greatest cooling of bronchial mucosa and lymph nodes (p < 0.001). The route of flush did not affect expression of RANTES, MCP-1, IL-8, IL-1beta, TNF-alpha or IL-6. However, expression of Gro was reduced 4-h post-preservation in all groups. Only AG/BA resulted in decreased IFN-gamma transcripts. These data show that, compared to AG or AG/RG, AG/BA flush results in the greatest cooling of lung compartments and down regulates lymph node expression of a cytokine and chemokine that have key roles in inflammation and immunity. These data suggest that pulmonary flush via AG/BA during donor harvest may be optimal to decrease the risk of early graft failure.

Retrograde vs. antegrade lung flush perfusion for graft preservation in clinical lung transplantation

Objectives: Reports by us and others on experimental studies have indicated a beneficial effect of retrograde lung flush perfusion on posttransplant outcome. We have developed a novel in-situ retrograde lung flush perfusion strategy and compared it with a common antegrade lung flush perfusion technique in clinical lung transplantation (LTx). Methods: 121 LTx procedures in 119 patients were consecutively included into the study from January 2002 to May 2003. All data were prospectively recorded. Antegrade (AP) donor lung perfusion was performed in 84, retrograde (RP) perfusion in 37 cases. Low-potassium dextrane glucose solution (LPDG) was used for lung preservation. Prostacycline (40 yg/l) was added to the flush solution. Main endpoints included ischemic time, PaO2/FiO2 ratio 2,4,8,12, and 24 hrs after arrival on the ICU, time to extubation, and 30-day mortality. Minor endpoints included indication for LTx, gender, age and donor/ recipient gender match. Results: There were no differences in total ischemic time in both groups with 310 98 vs. 305 102 min for right lungs and 373 91 vs. 369 93 min for left lungs in the AP and the RP group, respectively. Lung graft function (PaO2/FiO2 ratio) did not significantly differ among groups at any time point. However, there was a mild trend towards improved function in the RP group at the early time points. Posttransplant ventilation time was significantly lower in the RP group with 113 266 hrs vs. 212 328 hrs in the AP group (p 0.05). The mean length of posttransplant ICU stay was not different among groups (RP: 12.7 17.5 days vs. AP: 11.7 14.4 days). Perioperative mortality was 10 % in the RP group vs. 9% in the AP-cohort. Conclusions: This clinical study does not underline the previously, in experimental studies observed beneficial effect of retrograde lung flush perfusion for lung graft preservation compared to antegrade perfusion. Detailed investigations are warranted to determine these controversial results in human lung transplantation.

Improved lung function by means of retrograde flush in canine lung transplantation with non-heart-beating donors

Objective: Use of lungs from non-heart-beating donors would increase the pulmonary donor pool. The aim of this study was to evaluate the effects of retrograde flush in canine lung transplantation from non-heart-beating donors. Methods: Left lung transplantation was performed in 12 weight-matched pairs of dogs. Donors were killed without heparinization, left at room temperature for 2 hours, and then randomized into 2 groups. In group AF (n = 6) lung retrieval was performed after flushing the lung block with low potassium dextran glucose (50 mL/kg) solution through the pulmonary artery. In group AF+RF (n = 6) additional retrograde flushing (low potassium dextran glucose, 25 mL/kg) was performed through the left atrium before retrieval. Flushed solution was sampled at intervals to measure hemoglobin concentrations. The lungs were preserved at 4°C for 2 hours, and the left lung was implanted to the recipient being subjected to a total ischemic time of 5 hours. After left lung transplantation, the right pulmonary artery and main bronchus were ligated. Lung function, including arterial blood gas and pulmonary hemodynamics, was measured for 3 hours. For lung function study, statistical analyses were performed by using 1-way analysis of variance with repeated measures. Results: Group AF+RF had significantly better gas exchange and lower wet/dry weight ratio of the transplanted lung than group AF. Changes of hemoglobin concentration in the flushed solution indicated that additional retrograde flush could remove residual microthrombi after antegrade flush. Conclusions: This study supports the theory that additional retrograde flush improves lung function after lung transplantation by removing residual pulmonary microthrombi in the lungs of non-heart-beating donors.J Thorac Cardiovasc Surg 2003;125:901-6

Improvement of stallion spermatozoa preservation with alternative cryoprotectants for the Mangalarga Marchador breed

Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze, and (5) Gent + Gent Freeze. Extenders 1 and 2 showed higher values for total and progressive motility after thawing compared with extender 4 (P < .05). Extender 3 was in between 1 and 2 (P > .05), and extender 5 resulted in the lowest values (P < .05). The subpopulation of viable frozen-thawed sperm with high mitochondrial membrane potential and low intracellular calcium content was higher using extender 1 compared with extenders 3, 4, and 5 (P < .05) and higher in extender 2 compared with extenders 4 and 5 immediately after thawing (P < .05). After 1 hour of incubation, this subpopulation yielded the highest values in extender 2 (P < .05). Immediately after thawing, extender 1 yielded higher values for percentage of DFI and mean DFI than extenders 3, 4, and 5 (P < .05). Following 1 hour of incubation after thawing, sperm processed with extender 1 resulted in the highest values for percentage of DFI and mean DFI (P < .05). Using extender 2, mean DFI values were lower than those in extender 1 and higher than the extenders 3, 4, and 5 (P < .05). The study revealed that according to the examined sperm quality parameters, freezing extenders (extender 1, extender 2) using low concentrations of glycerol either combined with or without methylformamide were beneficial for cryopreservation of stallion epididymal sperm. For processing of stallion epididymal sperm, an extender containing milk proteins (extenders 1–4) for initial dilution after sperm harvesting is preferable to an extender including egg yolk (extender 5).

Surfactant function in lung transplantation after 24 hours of ischemia: Advantage of retrograde flush perfusion for preservation

Surfactant function was shown to be impaired in clinical and experimental lung transplantation. This study was designed to define the impact of retrograde flush perfusion on graft and surfactant function after an extended period of ischemia. Left lung transplantation was performed after 24 hours of graft ischemia in 12 pigs. In half of the grafts antegrade cold flush perfusion (Perfadex) was used for preservation. In the second group grafts were flushed in a retrograde fashion via the left atrium. Graft function was monitored for 7 hours after transplantation. Before transplantation (basal) and after 2 hours of reperfusion, bronchoalveolar lavage fluid was obtained. Minimal surface tension of bronchoalveolar lavage fluid was determined and the ratio of small and large surfactant aggregates was calculated. Lung water content was analyzed online in the reperfusion period. Right-sided heart failure developed in 2 animals of group 1 (antegrade perfusion) within 2 and 4.5 hours of reperfusion, respectively. All other pigs survived the observation period. PO(2)/FIO(2) (P =.001) and dynamic lung compliance (P =.001) were superior in retrogradely flushed grafts. A comparable increase of minimal surface tension was found after reperfusion in both groups. Small/large surfactant aggregate ratio after reperfusion (P =.03), as well as extravascular lung water content, was higher in the antegrade perfusion group. Retrograde flush perfusion for 24-hour lung preservation with low-potassium dextran (Perfadex) solution led to better initial graft function than the standard antegrade perfusion technique. A moderate impairment of surfactant function was found in both groups, which was more pronounced in the antegrade perfusion group.

Nitroglycerin alters alveolar type II cell ultrastructure after ischemia and reperfusion

Background: Although administration of nitric oxide (NO) has been suggested to reduce pulmonary reimplantation response, concerns remain about cytotoxic side effects. Methods: Using light and electron microscopy, we examined the effects of the NO donor nitroglycerin (NTG) (0.1 mg/ml) as a supplement to the preservation solution Celsior on the structural integrity of rat lungs after extracorporeal ischemia (4 hours at 10°C) and reperfusion (50 minutes) (IR). We performed evaluation in comparison with Celsior alone after IR using either standard antegrade perfusion through the pulmonary artery or retrograde perfusion through the left atrium as an alternative way to improve the preservation quality. Untreated, non-ischemic lungs served as controls ( n = 5 per group). We recorded respiratory and hemodynamic parameters during reperfusion. Tissue collection using systematic uniform random sampling was representative for the whole organ and allowed stereologic quantification of structures. Results: After IR, histochemistry revealed no breaks in the alveolo-capillary barrier and we detected no alveolar flooding. Edema formed in the peribronchovascular cuffs, of which the volume fraction was increased ( p = .008). Vasoconstriction of the smaller arteries accompanied antegrade flush, which occurred neither after administration of NTG nor after retrograde flush, as shown by immunostaining for α-smooth muscle actin. Treatment with NTG was associated with focal disintegration of Type II cells, which displayed edematous swelling of distinct cell compartments and lysis of mitochondria and cells. Nitroglycerin prevented alveolar collapse, which was increased in the other IR groups ( p = 0.013). We observed alterations in intra-alveolar surfactant components. Conclusion: These findings indicate pathologic effects of NTG treatment on alveolar epithelial integrity. Therefore, we suggest further critical evaluation of NTG/NO for therapeutic use in lung transplantation.

Stop-flow studies of distribution of filtration in rat lungs.

The stop-flow approach was used to investigate where filtration occurs in the pulmonary vasculature after elevation of left atrial pressure and aspiration of HCl. Rat lungs were perfused for 11 min at zero left atrial pressures, and then flow was stopped for 10 min and left atrial pressures were increased to 20 cmH2O. Thereafter, 3HOH was instilled into the air spaces, and the pulmonary vasculature was flushed by perfusing it from the pulmonary artery to left atrium (anterograde flush) or in the opposite direction (retrograde flush). Increases in fluorescein isothiocyanate (FITC)-dextran (molecular weight 2,000,000) indicated filtration, and these preceded increases in 3HOH after anterograde but not retrograde flushes. This suggests that some filtration occurred through vessels that were relatively venous compared with those through which 3HOH exchange had occurred. Filtration increased fivefold after instillation of 0.1 N HCl in isotonic saline into the air spaces before perfusion. Increases in Evans blue-labeled albumin concentrations were < 40% those of FITC-dextran, indicating loss from the vasculature, but increases in unlabeled albumin and FITC-albumin were comparable.

Intracellular EDEMA does not adversely affect the ability to cryopreserve intact hearts

The earliest ultrastructural manifestation of hypoxic cell injury is the presence of intracellular edema. Does this intracellular edema affect the ability to cryopreserve intact myocardium? To answer this guestion, a model for anoxia induced intracellular edema (IE) was designed based on clinical intraoperative myocardial preservation protocol. The aortas of 250 gm male Sprague-Dawley rats were cannulated and a retrograde flush of Plegisol at 8°C was infused over 90 sec. The hearts were excised and placed in a 28°C bath of Lactated Ringers for 1 h. The left ventricular free wall was then sliced and the myocardium was slam frozen. Control rats (C) were anesthetized, the hearts approached by median sternotomy, and the left ventricular free wall frozen in situ immediately after slicing. The slam frozen samples were obtained utilizing the DDK PS1000, which was precooled to -185°C in liguid nitrogen. The tissue was in contact with the metal mirror for a dwell time of 20 sec, and stored in liguid nitrogen until freeze dry processing (Lightfoot, 1990).

Miscellaneous Conditions of the Equine Head

Nutritional secondary hyperparathyroidism is associated with horses that are fed diets with an imbalance of calcium and phosphorus or diets containing oxalates. Clinical signs include a shifting lameness, with tenderness of the joints, reluctance to move, or a stiff, stilted gait. Some horses may have broadening of the nasal bones, enlargement of the maxilla and/or the mandible, or a ridge over the nasal bones. Diagnosis may include physical examination findings; analysis of feed; and clinical pathologic data, which may include normocalcemia, normal to increased serum phosphorus, and increased fractional excretion of phosphorus (normal FxP < 0.5%). The primary objective of therapy is to correct the nutritional calcium to phosphorus imbalance. Sialoliths are calcific concretions that develop in the salivary gland or duct. Therapy generally involves surgical removal, and associated complications are uncommon. Obstruction of the nasolacrimal duct occurs most commonly secondary to intraluminal foreign material, although congenital atresia may occur. Clinical signs include epiphora, conjunctivitis, and mucopurulent discharge. Foreign material is often removed by retrograde saline flush and atresia is resolved by surgery.

Observations on the loss of the cellular vestment surrounding superovulated sheep oocytes

Twenty-seven Merino and Merino × Border Leicester ewes were superovulated in the following manner 10 days after sponge insertion: Day 10 — 500 i.u. PMSG + 5 mg FSH-P; Day 11 — 300 i.u. PMSG + 4 mg FSH-P; Day 12 — 3 mg FSH-P + sponge removed. Twenty-four hours after sponge removal, 100 μg GnRH was administered. Tubal oocyte collections were performed at 18–21 (Group A), 21–25 (Group B) or 25–29 (Group C) hours after GnRH administration. Oocytes were recovered by a retrograde flush of the oviducts. Ovulation rates were 3.9 ± 1.7, 9.6 ± 1.5 and 11.9 ± 1.5 for Groups A, B and C respectively. The level of cellular vestment surrounding collected oocytes was noted. Oocytes were grouped into one of four categories: I — surrounded by an expanded cumulus/corona complex; II — surrounded by a corona only; III — devoid of all cellular vestments; IV — abnormal or degenerating oocytes. In Group A, 100% ( n = 5) oocytes were Type I. In Group B ( n = 44), 18.2% were Type I, 61.4% Type II, 18.2% Type III and 2.2% Type IV. In Group C ( n=70), 10% were Type II and 90% were Type III. Results indicate that ovulation had begun 18–21 h after GnRH, and was mostly complete by 25 h after GnRH. There was a marked rapid cumulus loss from superovulated sheep oocytes in the oviduct — within 4–6 h, oocytes were devoid of cellular vestments. The implications of these results are discussed.

Gene microinjection in bovine embryos facilitated by centrifugation

Twenty-seven Merino and Merino × Border Leicester ewes were superovulated in the following manner 10 days after sponge insertion: Day 10 — 500 i.u. PMSG + 5 mg FSH-P; Day 11 — 300 i.u. PMSG + 4 mg FSH-P; Day 12 — 3 mg FSH-P + sponge removed. Twenty-four hours after sponge removal, 100 μg GnRH was administered. Tubal oocyte collections were performed at 18–21 (Group A), 21–25 (Group B) or 25–29 (Group C) hours after GnRH administration. Oocytes were recovered by a retrograde flush of the oviducts. Ovulation rates were 3.9 ± 1.7, 9.6 ± 1.5 and 11.9 ± 1.5 for Groups A, B and C respectively.The level of cellular vestment surrounding collected oocytes was noted. Oocytes were grouped into one of four categories: I — surrounded by an expanded cumulus/corona complex; II — surrounded by a corona only; III — devoid of all cellular vestments; IV — abnormal or degenerating oocytes. In Group A, 100% (n = 5) oocytes were Type I. In Group B (n = 44), 18.2% were Type I, 61.4% Type II, 18.2% Type III and 2.2% Type IV. In Group C (n=70), 10% were Type II and 90% were Type III.Results indicate that ovulation had begun 18–21 h after GnRH, and was mostly complete by 25 h after GnRH. There was a marked rapid cumulus loss from superovulated sheep oocytes in the oviduct — within 4–6 h, oocytes were devoid of cellular vestments. The implications of these results are discussed.

Management of Patent Ductus Arteriosus in the Premature Infant: Indomethacin versus Ligation

A previous report from our institution analyzed the results of pharmacological (indomethacin) closure of patent ductus arteriosus (PDA) in 82 neonates. Closure was achieved in 54 patients. However, gastrointestinal complications occurred in 21, necrotizing enterocolitis in 13, and focal perforation in 8. Overall mortality in the indomethacin group was 40%. This paper compares the results of that pharmacological experience with our subsequent surgical experience with 86 low-birth-weight neonates for whom gestational age, size, illness, and mode of diagnosis were comparable. Mean weight at operation for this study was 1.1 kg; mean gestational age was 29.1 weeks. All infants required endotracheal-assisted ventilation for severe radiographic and clinical hyaline membrane disease. Range-gated Doppler study, retrograde flush aortography, and echocardiographic measurement of the ratio between the left atrium and the aortic root were used to confirm the diagnosis of PDA. Ligation was done in the neonatal intensive care unit using local anesthesia supplemented with morphine. Ventilation was controlled by an inhalation therapist; drug and blood administration were controlled by the infant's nurse. Surgical ligation was employed in all infants except for 7 in whom hemoclip ductal closure was chosen because of extreme instability, coagulopathy, or ductal perforation. There were no operative deaths. Surgical morbidity included ductal perforation (2 patients), wound infection (1), and phrenic nerve injury (1). Necrotizing enterocolitis occurred in 9 patients. The overall mortality was 17%. Patients with preoperative pneumothorax had a 32% overall mortality. Surgical closure of PDA can be accomplished safely with minimal anesthesia in an in-unit operating room. Hemoclip PDA closure can be used effectively in the occasional patient whose condition is unstable. Success is universal, and complications of indomethacin therapy are avoided.