A novel paracorporeal method for isolated rodent lung reperfusion.

BACKGROUND: The isolated perfused lung model is commonly used in small animals to study lung function after preservation and cold storage. Measurements of oxygenation, compliance, and capillary filtration coefficient (Kf) permit analysis of preservation solutions or modifications of these solutions. However, inter-investigator variability using different perfusates makes comparisons difficult. Whole blood perfusion more closely mimics the in vivo situation, but extracorporeal circulation may alter the physiologic integrity of the model. Paracorporeal support has been used, but this technique required mechanical ventilation of the support rodent and did not incorporate a method for determining Kf. We evaluated a less-invasive technique, of providing cross-circulatory syngeneic support, maintaining the ability to compute Kf. METHODS: Angiocatheters were inserted into both femoral arteries and one femoral vein of the support rat. The venous cannula was connected to the pulmonary artery of the ex vivo lung block to provide inflow. Pulmonary effluent blood from the lung block was collected via a left atrial cannula and returned to the support rat via the femoral artery. A separate, height-adjustable column was included in the circuit for measurement of Kf. RESULTS: Each support rat was used to sequentially perfuse three double-lung blocks. The inflow sample to each lung block was analyzed for pH, pO2, pCO2, and hematocrit to follow alterations in support rat physiology. There were no statistical differences in the pH, PO2, or hematocrit. No significant differences were noted in the pO2 of the pulmonary effluent blood or the Kf; analyzed to determine whether the sequence of reperfusion affected the pulmonary function assessment. CONCLUSIONS: The syngeneic support rat delivers constant pressure systemic venous blood at stable physiologic parameters to the ex vivo lung block. Recirculation of the perfusate through the support rat diminishes the need to pool blood from donors, detoxifies and deoxygenates pulmonary effluent blood, and permits examination of sequential lung blocks. This technique represents a hybrid model between isolated perfused and orthotopic transplant models, maintaining Kf determination, a sensitive indicator of reperfusion injury. This technique could be applicable to reperfusion injury models of other organs (using arterial inflow instead) and may permit increased standardization among investigators.

Platelet activating factor acetylhydrolase decreases lung reperfusion injury.

BACKGROUND: Ischemia-reperfusion injury involves free radical production, polymorphonuclear neutrophil chemotaxis/degranulation, and production of proteolytic enzymes, complement components, coagulation factors, and cytokines. Activated polymorphonuclear neutrophils, endothelial cells, and macrophages produce platelet activating factor, which further promotes these inflammatory reactions. The recently cloned plasma form of platelet activating factor-acetylhydrolase (PAF-AH) demonstrates antiinflammatory effects by degrading platelet activating factor. We evaluated the effects of PAF-AH in an isolated perfused rat lung model by adding it to the flush solutions or to the reperfusion blood. METHODS: Rat lungs were isolated, flushed with EuroCollins (EC) or University of Wisconsin (UW) solution, stored at 4 degrees C for 6 or 12 hours, and reperfused using a cross-circulating syngeneic support rat. During reperfusion, oxygenation, compliance, and capillary filtration coefficient were calculated. There were four groups in the study; group I (control) had no PAF-AH added, group II had PAF-AH added to the flush solution, group III had PAF-AH added to reperfusion blood, and group IV had PAF-AH added to both flush solution and reperfusion blood. RESULTS: After 6 hours of storage, oxygenation, compliance, and capillary filtration coefficient significantly improved for EC in group IV. For UW, oxygenation improved in group IV whereas compliance improved in groups II, III, and IV. After 12 hours of storage, compliance improved for EC in group IV and capillary filtration coefficient improved in groups III and IV. For UW, oxygenation and compliance improved in groups II and IV, whereas capillary filtration coefficient improved in group IV. CONCLUSIONS: Addition of PAF-AH to intracellular organ preservation solutions and to the blood reperfusate significantly improves postreperfusion oxygenation and compliance, and reduces lung capillary permeability.

Platelet activating factor modulates microvascular permeability through nitric oxide synthesis.

Biochemical signaling determines the specific action of vasomediators in the control of microvascular permeability and tone. We tested the hypothesis that nitric oxide (NO) synthesis is involved in the biochemical signaling pathway of platelet activating factor (PAF). The cheek pouch of anesthetized male Syrian hamsters was used as a microvascular model. Vessel diameter [expressed as the ratio of the experimental to the control (e/c) diameter, with control diameter normalized to 1] and extravasation of FITC-dextran 150 by integrated optical intensity (IOI) were determined using intravital fluorescent microscopy and computer-assisted digital image analysis. N-Nitro-L-arginine methyl ester (L-NAME) at 10(-5) and 10(-6) M and N-nitro-L-mono-methyl arginine (L-NMMA) at 10(-4) and 10(-5) M were used as inhibitors of NO synthase (NOS). Acetylcholine (ACh) and bradykinin were used as indirect indices of NOS activation. L-NAME and L-NMMA attenuated both ACh and bradykinin vasodilatory effects as well as the bradykinin-induced increase in vascular permeability. Topical PAF (10(-7) M) caused vasoconstriction (mean +/- SEM e/c ratio = 0.3 +/- 0.1) and increased IOI from a normalized baseline of 0 to 67.4 +/- 12.8. Topical administration of L-NAME produced differential effects on the series-arranged arterioles but had no effect on postcapillary venular permeability. L-NMMA did not influence the basal arteriolar diameter, but at 10(-5) M it caused a small increase in permeability (IOI = 14.3 +/- 4.2). In the presence of NOS inhibitors, PAF caused a reduced arteriolar constriction (e/c ratio = 0.6 +/- 0.1) relative to PAF alone. Both NOS inhibitors reduced the PAF-stimulated increase in vasopermeability. At 10(-5) M L-NMMA, the PAF-stimulated IOI mean value was 26.1 +/- 5.2, while at 10(-4) M L-NMMA the PAF-stimulated IOI was 15.2 +/- 2.6 compared to 10(-7) M PAF (67.4 +/- 12.8). These results support our hypothesis that NO synthesis is a step in the biochemical signaling pathway of the postcapillary cellular responses to PAF.