Alteration of Neuropeptides in the Lung Tissue Correlates Brain Death-Induced Neurogenic Edema

Alteration of Neuropeptides in the Lung Tissue Correlates Brain Death-Induced Neurogenic Edema Anne Barklin, MD, PhD,a Elvar Theodorsson, DMSc,b Stig S. Tyvold, MD,c Anders Larsson, DMSc,a Asger Granfeldt, MD,a Erik Sloth, MDSc,a and Else Tonnesen, DMScb Background: Increased intracranial pressure induces neurogenic pulmonary edema (NPE), potentially explaining why only lungs from less than 20% of brain dead organ donors can be used for transplantation. This study investigated the underlying mechanisms of NPE, focusing on neuropeptides, which potently induce vasoconstriction, vasodilatation, and neurogenic inflammation. Methods: Brain death was induced in 10 pigs by increasing the intracranial pressure. Eight additional pigs served as controls. Neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and substance P were analyzed in plasma, bronchoalveolar lavage (BAL) fluid, and homogenized lung tissue 6 hours after brain death. Pulmonary oxygen exchange was estimated using partial pressure of arterial oxygen (PaO2)/fraction of inspired oxygen (FIO2), and pulmonary edema by wet/dry weight ratio. Results: Brain death induced a decrease in PaO2/FIO2 (p ⬍ 0.001) and increased the wet/dry weight of both apical (p ⫽ 0.01) and basal lobes (p ⫽ 0.03). NPY and CGRP concentrations were higher in the BAL fluid of brain-dead animals compared with controls (p ⫽ 0.02 and p ⫽ 0.02) and were positively correlated with the wet/dry weight ratio. NPY content in lung tissue was lower in brain-dead animals compared with controls (p ⫽ 0.04) and was negatively correlated with the wet/dry weight ratio. There were no differences in substance P concentrations between the groups. Conclusion: NPY was released from the lung tissue of brain-dead pigs, and its concentration was related to the extent of pulmonary edema. NPY may be one of several crucial mediators of neurogenic pulmonary edema, raising the possibility of treatment with NPY-antagonists to increase the number of available lung donors. J Heart Lung Transplant 2009;28:725–32. Copyright © 2009 by the International Society for Heart and Lung Transplantation.

Lung transplantation is often the only treatment option for patients with end-stage pulmonary disease. The severe shortage of donor lungs is a major limiting factor, however, and many patients die while on the waiting list for transplantation.1 The lack of organs particularly affects patients with lung disease, because less than 20% of organ donors possess lungs suitable for transplantation.1,2 Therefore, it is important to focus on the underlying mechanisms of brain death-induced lung

From the aDepartment of Anesthesiology, Aarhus University Hospital, Aarhus, Denmark; bIKE/Clinical Chemistry, University Hospital, Linköping, Sweden; and cDepartment of Circulation and Medical Imaging, The Norwegian University of Science and Technology (NTNU), Trondheim, Norway. Submitted December 1, 2008; revised February 23, 2009; accepted April 7, 2009. Funding: Aarhus University Research Foundation, The Danish Research Council, Holger og Ruth Hesses Mindefond. Reprint requests: Anne Barklin, Department of Anesthesiology, Aarhus Universital Hospital, Noerrebrogade 44, Building 1C, 1st, 8000 Aarhus C, Denmark. Telephone: ⫹45-8949-2852. E-mail: Barklin@ki. au.dk Copyright © 2009 by the International Society for Heart and Lung Transplantation. 1053-2498/09/$–see front matter. doi:10.1016/ j.healun.2009.04.008

dysfunction to improve the management of brain dead donors to increase the number of transplantable lungs. Neurogenic pulmonary edema (NPE) is a serious complication that occurs immediately after intracranial injury and leads to impaired oxygenation.3,4 NPE was first described more than 100 years ago, but the mechanism behind NPE is still debated. It is believed to be through increased pulmonary capillary hydrostatic pressure induced by catecholamines, or increased capillary permeability elicited by catecholamines directly or by inflammatory mediators.3,5 Sympathetic nerve stimulation increases pulmonary vascular permeability even after depletion of intraneuronal catecholamines,6 indicating that other sympathetic mediators might be responsible for NPE. Neuropeptide Y (NPY) is released from sympathetic nerves and is a potent vasoconstrictor.7 NPY is increased in the NPE fluid of rats,8 and NPY-antagonist treatment reduces capillary permeability.9 Thus, NPY may be one of several important mediators in NPE. We are not aware of any previous studies investigating the role of NPY in the lungs of a large-animal model. Substance P,10 which is released from sensory neurons that innervate the bronchial epithelium, is a potent inducer of neurogenic inflammation in the airways.11,12 725

726

Barklin et al

Of the other sensory neuropeptides in the lungs, calcitonin gene-related peptide (CGRP) is the most abundant.13 CGRP is primarily considered as a modulator rather than a direct mediator of neurogenic inflammation.13 CGRP is the most potent vasodilator identified to date.14 Whether substance P and CGRP are released from the lungs during or after brain death remains unknown. The aim of this study was to further investigate the mechanisms underlying NPE, focusing on neuropeptides that potently induce vasoconstriction (NPY), vasodilatation (CGRP), or neurogenic inflammation (substance P). We hypothesized that brain death induces release of NPY, substance P, and CGRP, detected as elevated concentrations in the plasma and bronchoalveolar lavage (BAL) fluid and as reduced concentrations in homogenized lung tissue. Second, we hypothesized that NPY, substance P, and CGRP concentrations in the lungs were correlated with pulmonary edema. We tested this in a large-animal model of brain death.

The Journal of Heart and Lung Transplantation July 2009

group. A steel introducer needle from a 22 gauge Venflon (Becton Dickinson, Franklin Lakes, NJ, USA) was introduced subdurally into the posterior hole to measure intracranial pressure (ICP). The balloon was inflated with saline (1 ml/4 min) and, after a median of 64 minutes (range, 52–108 minutes) corresponding to a balloon inflation volume of 16 ml (range, 13–27 ml), the cerebral perfusion pressure became negative, meeting the diagnostic criteria for brain death.17 Immediately afterwards, the balloon was filled to a total volume of 30 ml. To avoid brain stem reflex-induced myoclonias, cisatracurium (0.6 mg/kg) was administered before the final bolus in the balloon. Fluid Resuscitation Ringer’s acetate solution was infused at a rate of 10 ml/kg. To prevent hypotension, 500 ml of Voluven (Fresenius Kabi, Bad Homburg, Germany) was administered 30 minutes after brain death. In the sham-operated animals, the Ringer’s acetate infusion was stopped while Voluven was administered. Mean arterial pressure was maintained above 60 mm Hg by intravenous bolus infusions of 300 mL of Ringer’s acetate solution, and repeated if necessary. No vasopressors or inotropic drugs were used. The BD pigs presented diabetes insipidus, and hourly urine volumes of more than 200 mL were substituted by Ringer’s acetate solution the following hour.

MATERIALS AND METHODS The study was approved by the Danish Inspectorate of Animal Experimentation. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources and published by the National Institutes of Health, Publication No. 86-23, revised 1996). The study used 18 female Danish Landrace pigs (mean weight, 29 kg; range, 28 –33 kg). Ten pigs comprised the brain-dead group (BD), and eight pigs the sham-operated group (control). Anesthesia was induced by intravenous ketamine (10 ␮g/kg) and midazolam (0.5 mg/ kg), and was maintained by continuous infusion of fentanyl (60 ␮g/kg/hour) and midazolam (6 mg/kg/ hour). The pigs were tracheotomized, and the lungs were ventilated with a tidal volume of 8 ml/kg/min, positive end-expiratory pressure of 5 cm H2O, and fraction of inspired oxygen (FIO2) of 0.4. The rate was adjusted to maintain a partial pressure of carbon dioxide (PCO2) at between 5.5 and 6.5 kPa. A Swan-Ganz catheter (Edwards Lifesciences, Irvine, CA) was inserted through the right jugular vein into the pulmonary artery. Cardiac output (CO) was continuously monitored (Edwards Vigilance Monitor, Edwards Lifescience). After BAL of the left lung and evisceration of the right lung 6 hours after brain death, the animals were killed by evisceration of the heart.

Bronchoalveolar Lavage Single-cycle BAL following the described by Peterson.20 Saline (60 ml) was introduced into the left lung, and a mean 24.5 ⫾ 6.6 (SD) ml was immediately withdrawn. The first 10 ml was discarded, and the remainder was centrifuged (1,000g) for 5 minutes. The supernatant was frozen immediately thereafter.

Induction of Brain Death Two holes were drilled in the cranium, as previously described.15,16 A 20F Foley catheter was inserted, and the balloon was placed in the epidural space in the BD

Neuropeptides Neuropeptide Y (NPY-LI) was analyzed using anti-serum 2151 raised in rabbits against conjugated human/rat NPY. The anti-serum cross-reacts equally with porcine

Wet/Dry Weight Ratio Pulmonary edema was assessed by measuring the wet/ dry weight ratio.18,19 The apical and basal lobes of the right lung were weighed immediately and after drying in an oven at 65°C. Blood Samples Blood gases were sampled every 15 minutes until 60 minutes after brain death, and hourly thereafter. They were analyzed by an ABL 700 Unit (Radiometer, Copenhagen, Denmark). Blood for detection of neuropeptides was sampled at baseline, at the time of brain death, and 15, 30, 60, 120, 240, and 360 minutes after brain death.

The Journal of Heart and Lung Transplantation Volume 28, Number 7

and human/rat NPY, but not with substance P (SP), CGRP, pancreatic polypeptide, or vasoactive intestinal peptide. The radioligand was 125I-human/rat NPY purified by high-performance liquid chromatography (HPLC), and the standard was porcine NPY. The detection limit of the assay was 1.4 pmol/liter (half maximal inhibitory concentration was 24 pmol/liter), and the intra- and interassay coefficients of variation were 5% and 8%, respectively.21 Substance P (SP-LI) was analyzed using anti-serum SP2,22 which reacts with SP and SP sulfoxide, but not with other tachykinins. The detection limit was 10 pmol/liter. Intra- and interassay coefficients of variation were 7% and 11%, respectively.22 Calcitonin gene-related peptide (CGRP-LI) was analyzed using anti-serum CGRPR8 raised in rabbit against conjugated rat CGRP. HPLC-purified 125I-Histidyl rat CGRP was used as a radioligand and rat CGRP was the standard. The detection limit of the assay for rat CGRP is 9 pmol/L, and the assay cross-reactivity with substance P, neurokinin A, neurokinin B, neuropeptide K, gastrin, neurotensin, bombesin, neuropeptide Y, and calcitonin was less than 0.01%. Cross-reactivity with human CGRP-␣ and CGRP-␤ was 93% and 24%, respectively, and 100% and 120% with rat CGRP-␣ and CGRP-␤, respectively. Intra- and interassay coefficients of variation were 8% and 14%, respectively. Tissue and Plasma Sample Extraction Lung tissue was cut into small pieces, and 10 mL of 1 mol/liter acetic acid was added to each gram of tissue (Merck, Darmstadt, Germany). The tissues were boiled for 10 minutes and homogenized using a polytron (CAT X520D, Zipperer, Staufen, Germany) and centrifuged at 1,500g for 10 minutes at 4°C. The supernatants were lyophilized and stored at ⫺70°C until neuropeptide analysis. Plasma samples were extracted before analysis on C18-reverse-phase cartridges, as described elsewhere.23 Echocardiography Transthoracic echocardiography was performed 6 times: at baseline, at the time of brain death, and 30, 60, 120, and 360 minutes after brain death using a Vivid 7 ultrasound machine equipped with a 2.5-MHz matrix probe (GE Healthcare, Horten, Norway). A midpapillary parasternal short-axis view of the left ventricle was obtained, and multiple cine loops were digitally stored for off-line analysis. The split-screen feature was used, enabling simultaneous display of a reference image and the real-time image. The left ventricular end-diastolic area (LVEDA) and end-systolic area (LVESA) of 1 cine loop were traced using dedicated software (EchoPac; GE Healthcare, Horten, Norway). On the same image, the eccentricity index was calculated. A high eccentric-

Barklin et al

727

ity index represents a flatter intraventricular septum, indicating right ventricular overload.24 Ejection fraction was calculated from the Teichholz formula using the anatomical m-mode to ensure that the m-mode line was perpendicular to the septum and posterior wall. Statistics Differences between the groups were compared using unpaired t-tests if the data were normally distributed and the Mann-Whitney test if not. Changes in plasma neuropeptides over time within and between the groups were compared using analysis of variance for repeated measurements. Differences between the 2 groups regarding hemodynamics and echocardiography data were evaluated by the t-test for values at baseline, 6 hours after brain death, and maximal values. Spearman’s ␳ was calculated to describe the correlations between NPY, CGRP, and extent of edema. Analyses were performed using Stata 9.2 software (StataCorp LP, College Station, TX). Two-tailed values of p ⬍ 0.05 were considered statistically significant. RESULTS Cardiac arrest occurred in 1 pig at 180 minutes after brain death. Partial Pressure of Arterial Oxygen/Fraction of Inspired Oxygen The initial partial pressure of arterial oxygen (PaO2)/ fraction of inspired oxygen (FIO2) ratio did not differ between the 2 groups, but it was significantly lower after pigs after brain death compared with the controls (p ⬍ 0.001; Figure 1).

Figure 1. Mean data are shown with the standard error (range bars) for the partial pressure of arterial oxygen (PaO2)/fraction of inspired oxygen (FIO2) in the brain dead (black circles) and control (white circles) pigs. Differences were analyzed by analysis of variance for repeated measures. BL, baseline; BD, brain death. #p ⬍ 0.001.

728

Barklin et al

The Journal of Heart and Lung Transplantation July 2009

Figure 2. Mean plasma levels of neuropeptide Y (NPY) in brain dead (black dots) and control (white dots) pigs are shown with the standard error (range bars). BL, baseline; BD, brain death. Differences between groups were analyzed using analysis of variance for repeated measurements. #p ⬍ 0.001.

Pulmonary Edema The water content was higher, as indicated by higher median (range) wet/dry weight ratios, in the BD pigs compared with controls in apical (5.57 [5.14 – 6.64] vs 5.23 [4.22–5.33]; p ⫽ 0.01), as well as basal lobes (5.36 [5.14 – 6.35] vs 4.95 [4.58 –5.25]; p ⫽ 0.03).

Figure 4. Neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and substance P (SP) in homogenized lung tissue 6 hours after brain death in brain dead (BD) and control (C) pigs. Mann-Whitney test, *p ⬍ 0.05.

Neuropeptides

Figure 3. Neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and substance P (SP) in bronchioalveolar lavage (BAL) fluid 6 hours after brain death in brain dead (BD, black circles) and control (C, white circles) pigs. Mann-Whitney test,*p ⬍ 0.05.

Plasma concentrations of NPY increased significantly over time in response to brain death (p ⬍ 0.001), and between BD and control animals (p ⬍ 0.001; Figure 2). No differences were detected within or between groups regarding plasma-CGRP or plasma-substance P. Median (range) basal levels in pmol/l of CGRP were 27 (19 –34) in BD and 24 (23–29) in controls and basal levels of substance P were 27 (7– 42) in BD and 35 (10 – 42) in controls. The concentration of NPY and CGRP in BAL-fluid was higher in BD animals compared with controls (p ⫽ 0.02 for both; Figure 3).

The Journal of Heart and Lung Transplantation Volume 28, Number 7

NPY was significantly lower in basal lobe lung tissue from BD animals compared with controls (p ⫽ 0.04). No differences were observed between the apical lobes of the BD and control pigs (Figure 4). Neuropeptides in Correlation With Pulmonary Edema The wet/dry weight ratio of the lower lobe was significantly correlated with NPY in lung tissue (r ⫽ ⫺0.7, p ⫽ 0.007), and this result could be confirmed when only testing the BD animals (r ⫽ ⫺0.8, p ⫽ 0.01; Figure 5). Also significantly correlated with the wet/dry weight ratio were NPY (r ⫽ 0.5, p ⫽ 0.03) and CGRP (r ⫽ 0.5, p ⫽ 0.04) levels in the BAL fluid. Hemodynamics and Echocardiography Heart rate, mean arterial pressure, cardiac output, and pulmonary arterial pressure increased significantly (p ⬍ 0.001) after induction of brain death. Central venous pressure (CVP) did not change and was not different between the two groups at any time. The end-diastolic left ventricle inner diameter and area decreased markedly at the time of brain death induction and after 30 minutes, and then returned to

Barklin et al

729

baseline levels after 1 hour (Table 1). The ejection fraction increased significantly at the time of brain death induction but decreased over the next hour to baseline. The stroke volume remained unchanged and did not differ between groups. No differences in systolic and diastolic eccentricity indexes were detected at any time points, indicating no volume or pressure overload of the right ventricle. Fluid Resuscitation To maintain mean arterial pressure above 60 mm Hg, boluses of Ringer’s acetate solution were repeatedly infused into BD pigs (median, 600 ml; range, 0 –2600 ml; Figure 4). Total urine output during the course of the experiment was significantly higher in BD animals (3141 ⫾ 218 ml) than in controls (675 ⫾ 88 ml), and the urine sodium value became significantly lower in BD animals (54 ⫾ 5.3 mmol/liter) vs controls (139 ⫾ 16 mmol/liter) at the end of the experiment. There were no differences in the cumulative fluid balance (infusion minus output; BD: 1677 ⫾ 261, control: 1792 ⫾ 118; p ⫽ 0.70) (Figure 6) or hematocrit between groups (BD: 25.6 ⫾ 0.6, control: 25.1 ⫾ 0.3; p ⫽ 0.84) at the end of the experiment.

Figure 5. Neuropeptides in bronchioalveolar lavage (BAL) related to the wet/dry (W/D) weight of lungs from brain dead and control pigs. NPY, neuropeptide Y; CGRP, calcitonin gene-related peptide; SP, substance P. Correlation analyzed by Spearman rank correlation.

730

Barklin et al

The Journal of Heart and Lung Transplantation July 2009

Table 1. Hemodynamic and Echocardiographic Data Variable Intracranial pressure, mm Hg Brain dead Heart rate, beats/min Brain dead Control MAP, mm Hg Brain dead Control Cardiac output Brain dead Control Mean PAP, mm Hg Brain dead Control CVP, mm Hg Brain dead Control Ejection fraction, % Brain dead Control Stroke volume, ml Brain dead Control LVID end-diastolic, mm Brain dead Control Systolic eccentricity index Brain dead Control Diastolic eccentricity index Brain dead Control

Baseline

BD

30 min

60 min

120 min

360 min

p-value

14 ⫾ 3

325 ⫾ 31

223 ⫾ 89

192 ⫾ 94

169 ⫾ 94

127 ⫾ 57

⬍0.05

61 ⫾ 11 65 ⫾ 11

235 ⫾ 17 65 ⫾ 11

161 ⫾ 38 78 ⫾ 21

128 ⫾ 21 72 ⫾ 9

110 ⫾ 20 66 ⫾ 6

105 ⫾ 41 67 ⫾ 14

⬍0.05a,b ⬍0.05

78 ⫾ 28 87 ⫾ 9

187 ⫾ 37 83 ⫾ 9

68 ⫾ 14 87 ⫾ 15

74 ⫾ 13 80 ⫾ 12

81 ⫾ 18 76 ⫾ 8

77 ⫾ 14 72 ⫾ 11

⬍0.05a ⬍0.05

2.8 ⫾ 0.5 2.9 ⫾ 0.5

6.2 ⫾ 1.8 2.9 ⫾ 0.6

6.5 ⫾ 1.5 3.2 ⫾ 0.5

7.2 ⫾ 1.8 3.3 ⫾ 0.5

5.3 ⫾ 0.8 3.1 ⫾ 0.4

4.9 ⫾ 0.9 2.7 ⫾ 0.5

⬍0.05a,b ⬍0.05

16 ⫾ 2 15 ⫾ 2

31 ⫾ 6 15 ⫾ 2

25 ⫾ 4 16 ⫾ 1

23 ⫾ 5 17 ⫾ 2

19 ⫾ 5 17 ⫾ 1

19 ⫾ 4 18 ⫾ 2

⬍0.05a ⬍0.05

8⫾3 7⫾2

8⫾2 7⫾2

8⫾4 7⫾1

10 ⫾ 4 10 ⫾ 2

8⫾2 9⫾1

8⫾2 9⫾1

⬍0.05 ⬍0.05

52 ⫾ 9 58 ⫾ 5

80 ⫾ 12 61 ⫾ 6

70 ⫾ 14 59 ⫾ 9

65 ⫾ 12 58 ⫾ 5

61 ⫾ 8 60 ⫾ 6

62 ⫾ 11 53 ⫾ 13

⬍0.05a ⬍0.05

37 ⫾ 9 47 ⫾ 9

39 ⫾ 17 46 ⫾ 11

36 ⫾ 12 44 ⫾ 6

50 ⫾ 17 46 ⫾ 11

48 ⫾ 10 51 ⫾ 14

48 ⫾ 13 40 ⫾ 12

⬍0.05 ⬍0.05

40 ⫾ 4 42 ⫾ 3

34 ⫾ 7 41 ⫾ 4

35 ⫾ 5 41 ⫾ 3

42 ⫾ 7 42 ⫾ 4

42 ⫾ 4 43 ⫾ 4

42 ⫾ 5 40 ⫾ 4

⬍0.05a ⬍0.05

0.98 ⫾ 0.08 0.97 ⫾ 0.05

0.91 ⫾ 0.39 1.04 ⫾ 0.18

0.92 ⫾ 0.16 1.01 ⫾ 0.07

0.96 ⫾ 0.05 1.00 ⫾ 0.07

1.01 ⫾ 0.04 0.98 ⫾ 0.05

0.97 ⫾ 0.05 0.97 ⫾ 0.05

⬍0.05 ⬍0.05

1.06 ⫾ 0.05 1.08 ⫾ 0.16

0.98 ⫾ 0.04 1.09 ⫾ 0.10

1.02 ⫾ 0.08 1.05 ⫾ 0.07

1.01 ⫾ 0.07 0.99 ⫾ 0.07

1.03 ⫾ 0.05 1.01 ⫾ 0.08

1.01 ⫾ 0.05 0.99 ⫾ 0.03

⬍0.05 ⬍0.05

BD, brain death; CVP, central venous pressure; LVID, left ventricle inner diameter; MAP, mean arterial pressure; PAP, pulmonary artery pressure. a Maximum values between the 2 groups. b Differences in end-baseline values between the 2 groups.

Discussion In this model, brain death induced pulmonary edema, which could not be explained by fluid overloading or cardiac dysfunction. Furthermore, the extent of pulmonary edema was positively correlated with the concentration of NPY and CGRP in BAL fluid and negatively correlated with the NPY concentration in lung tissue. We did not identify a systemic or pulmonary substance P response after brain death. Pulmonary edema is strongly dependent on fluid resuscitation and cardiac function. We maintained a similar total fluid balance across the 2 groups, and the central venous pressure, hematocrit, and the inner diameter of the left ventricle did not indicate any hypervolemia in the BD group. Interestingly, echocardiography did not reveal any signs of myocardial failure after brain death, which is not consistent with the findings of other studies.25,26 This is likely due to the sustained pre-load and after-load conditions of the heart

in this model.26 We identified no differences between the 2 groups with respect to any echocardiographic parameters at 2 or 6 hours after brain death, indicating that the pulmonary edema was not caused by cardiac dysfunction. The levels of NPY in the BAL fluid were positively correlated with lung water content. This finding confirms the work of Hawdy et al8 in rats, suggesting that NPY probably has an important effect in NPE development. Interestingly, NPY was significantly lower in the lung tissue from BD pigs compared with controls, indicating that NPY was released from the nerve endings in response to brain death. NPY is synthesized in the nerve nucleus and reaches the nerve endings via axonal transport, about 1 to 2 mm/hour,27 where it is stored in dense core vesicles until release.28 This finding indicates that NPY is released in the lungs and elicits its effect directly, and not as a systemic vasoconstrictor. NPY directly increases endothelial permeability, as was

The Journal of Heart and Lung Transplantation Volume 28, Number 7

recently confirmed in an in vitro model of the aortic endothelium, but only in hypoxic and not in normoxic conditions.29 In all BD animals in the present study, PaO2 was always above 15 kPa, and therefore a direct effect on the capillary endothelium is unlikely. It is probable that NPY in the lungs acts primarily as a post-capillary vasoconstrictor. Constriction of pre-capillaries may protect connected capillaries against high pulmonary artery pressure. According to the Starling equilibrium, this in turn would reduce the capillary hydrostatic pressure, and thus limit edema formation. The increased CGRP in the BAL fluid suggests that this neuropeptide is involved in NPE development. We have not yet investigated whether this effect is direct and linked to capillary leakage or indirect and associated with pre-capillary vasodilation, resulting in increased capillary hypostatic pressure. However, because pulmonary artery pressure increases dramatically at the time of herniation of the brain stem, a local

Barklin et al

731

precapillary vasodilatory effect associated with CGRP might theoretically increase the hydrostatic pressure and capillary-alveolar fluid leakage. Substance P is a potent inducer of inflammation.11 In the present study, the lack of substance P release in the blood, airways, or lungs indicates that NPE is probably not of inflammatory origin. However, as suggested by others and confirmed by our findings demonstrating increased NPY and CCPG, it may be have a vascular genesis. The present study has some limitations in respect to our attempts to extrapolate to the clinical situation. We use a standardized model in which brain death occurs after approximately 1 hour of increasing intracranial pressure, and continued the experiment until 6 hours after brain death. In clinical scenarios, the time during which intracranial pressure increases and leads to brain death is often longer, as is the time from brain death until organ harvest. Furthermore, our model is based solely on an increase in ICP, with no previous intracranial damage or bleeding. In conclusion, brain death induced pulmonary edema as assessed by a rapid decrease in pulmonary oxygen exchange and an increase in pulmonary water content, without evidence of myocardial dysfunction. NPY was released from the lungs in response to brain death and was correlated with the extent of pulmonary edema. We speculate that NPY antagonist treatment of the BD organ donor may increase the quantity and quality of lungs available for transplantation. The authors thank Lene Vestergård, Violetta Ashby, Mike B. Morgan, and Henrik Sørensen at Aarhus University and Bibbi Mårdh at Linköping University for their practical assistance.

REFERENCES

Figure 6. (Top) Fluid infusion per hour and cumulative fluid balance (CFB) in brain dead and control pigs. (Bottom) Urine output per hour. Mean and stand error (range bars). BL, baseline; BD, brain death.

1. Snell GI, Griffiths A, Levvey BJ, Oto T. Availability of lungs for transplantation: exploring the real potential of the donor pool. J Heart Lung Transplant 2008;27:662–7. 2. Trulock EP. Lung transplantation. Am J Respir Crit Care Med 1997;155:789 – 818. 3. Baumann A, Audibert G, McDonnell J, Mertes PM. Neurogenic pulmonary edema. Acta Anaesthesiol Scand 2007;51:447–55. 4. Fontes RB, Aguiar PH, Zanetti MV, Andrade F, Mandel M, Teixeira MJ. Acute neurogenic pulmonary edema: case reports and literature review. J Neurosurg Anesthesiol 2003;15:144 –50. 5. Avlonitis VS, Fisher AJ, Kirby JA, Dark JH. Pulmonary transplantation: the role of brain death in donor lung injury. Transplantation 2003;75:1928 –33. 6. Sakakibara H, Hashiba Y, Taki K, Kawanishi M, Shimada Y, Ishikawa N. Effect of sympathetic nerve stimulation on lung vascular permeability in the rat. Am Rev Respir Dis 1992;145:685–92. 7. Groneberg DA, Folkerts G, Peiser C, Chung KF, Fischer A. Neuropeptide Y (NPY). Pulm Pharmacol Ther 2004;17:173– 80. 8. Hamdy O, Nishiwaki K, Yajima M, et al. Presence and quantification of neuropeptide Y in pulmonary edema fluids in rats. Exp Lung Res 2000;26:137– 47. 9. Hirabayashi A, Nishiwaki K, Shimada Y, Ishikawa N. Role of neuropeptide Y and its receptor subtypes in neurogenic pulmonary edema. Eur J Pharmacol 1996;296:297–305.

732

Barklin et al

10. McDonald DM, Bowden JJ, Baluk P, Bunnett NW. Neurogenic inflammation. A model for studying efferent actions of sensory nerves. Adv Exp Med Biol 1996;410:453– 62. 11. Barnes PJ. Neurogenic inflammation in the airways. Respir Physiol 2001;125:145–54. 12. Coleridge JC, Coleridge HM. Afferent vagal C fibre innervation of the lungs and airways and its functional significance. Rev Physiol Biochem Pharmacol 1984;99:1–110. 13. Dakhama A, Larsen GL, Gelfand EW. Calcitonin gene-related peptide: role in airway homeostasis. Curr Opin Pharmacol 2004; 4:215–20. 14. Wimalawansa SJ. Calcitonin gene-related peptide and its receptors: molecular genetics, physiology, pathophysiology, and therapeutic potentials. Endocr Rev 1996;17:533– 85. 15. Barklin A, Larsson A, Vestergaard C, et al. Does brain death induce a pro-inflammatory response at the organ level in a porcine model? Acta Anaesthesiol Scand 2008;52:621–7. 16. Barklin A, Larsson A, Vestergaard C, et al. Insulin alters cytokine content in two pivotal organs after brain death: a porcine model. Acta Anaesthesiol Scand 2008;52:628 –34. 17. Wijdicks EF. The diagnosis of brain death. N Engl J Med 2001; 344:1215–21. 18. Wittwer T, Franke UF, Fehrenbach A, et al. Donor pretreatment using the aerosolized prostacyclin analogue iloprost optimizes post-ischemic function of non-heart beating donor lungs. J Heart Lung Transplant 2005;24:371– 8. 19. Steinberg J, Halter J, Schiller H, et al. Chemically modified tetracycline prevents the development of septic shock and acute respiratory distress syndrome in a clinically applicable porcine model. Shock 2005;24:348 –56.

The Journal of Heart and Lung Transplantation July 2009

20. Peterson BT, Griffith DE, Tate RW, Clancy SJ. Single-cycle bronchoalveolar lavage to determine solute concentrations in epithelial lining fluid. Am Rev Respir Dis 1993;147:1216 –22. 21. Theodorsson-Norheim E, Hemsen A, Lundberg JM. Radioimmunoassay for neuropeptide Y (NPY): chromatographic characterization of immunoreactivity in plasma and tissue extracts. Scand J Clin Lab Invest 1985;45:355– 65. 22. Brodin E, Lindefors N, Dalsgaard CJ, Theodorsson-Norheim E, Rosell S. Tachykinin multiplicity in rat central nervous system as studied using antisera raised against substance P and neurokinin A. Regul Pept 1986;13:253–72. 23. Theodorsson-Norheim E, Hemsen A, Brodin E, Lundberg JM. Sample handling techniques when analyzing regulatory peptides. Life Sci 1987;41:845– 8. 24. Ryan T, Petrovic O, Dillon JC, Feigenbaum H, Conley MJ, Armstrong WF. An echocardiographic index for separation of right ventricular volume and pressure overload. J Am Coll Cardiol 1985;5:918 –27. 25. Lyons JM, Pearl JM, McLean KM, et al. Glucocorticoid administration reduces cardiac dysfunction after brain death in pigs. J Heart Lung Transplant 2005;24:2249 –54. 26. Szabo G. Physiologic changes after brain death. J Heart Lung Transplant 2004;23(9 suppl):S223– 6. 27. Roos J, Kelly RB. Preassembly and transport of nerve terminals: a new concept of axonal transport. Nat Neurosci 2000;3:415–7. 28. Hokfelt T, Bartfai T, Bloom F. Neuropeptides: opportunities for drug discovery. Lancet Neurol 2003;2:463–72. 29. Nan YS, Feng GG, Hotta Y, et al. Neuropeptide Y enhances permeability across a rat aortic endothelial cell monolayer. Am J Physiol Heart Circ Physiol 2004;286:H1027–33.