Effects of Ventilation on Lung Function after Normothermic Ischemia

Introduction

The pathogenesis of postreperfusion lung dysfunction remains unclear despite extensive research. The state of the lung during ischemia, which seems to have mechanical consequences but is in fact a cellular effect, was the subject of considerable scientific interest 25 years ago. However, there were no further reports in the literature until 1992. ¹ Early studies concluded that a collapsed lung showed better pulmonary preservation in both allo-transplantation and in-situ warm ischemia-reperfusion models. ^{2 –4} Puskas and colleagues ¹ suggested that hyper-inflation provided better protection due to pulmonary vasodilatation during flush, increased surfactant release, and prevention of alveolar collapse. Almost all experi-mental studies have been on late postischemic function and there are few recent studies on immediate postischemic lung function and histology, in which early graft dys-function can be observed. This experimental study was carried out to compare the effects of atelectasis and hyperinflation on immediate postischemic lung function and architecture after normothermic ischemia.

Materials and Methods

Thirty Sprague-Dawley rats, weighing 270 to 330 g, were divided into 3 groups; the 2 experimental groups were subjected to 60 minutes of normothermic ischemia. The lungs were atelectatic in 10 (group A), they were hyper-inflated to a pressure of 10 cm H₂O in 10 (group B), and 10 rats served as controls (group C). All animals received human 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 the Use of Laboratory Animals” prepared by the National Academy of Science and published by the National Institutes of Health (NIH publication no. 85–23, revised 1985).

All rats were given 6 mg ketamine (Ketalar; Parke-Davis, Morris Plains, NJ, USA), 5mg sodium thiopental (Pentothal; Abbot, North Chicago, IL, USA), and 0.06 mg atropine (Galen Drugs, Istanbul, Turkey), intra-peritoneally. After tracheotomy, a 5F dilatator cannula (American Edwards Laboratories AHS del Caribe, Inc., Anasco, Puerto Rico) was inserted into the trachea. Mechanical ventilation was established by a rat ventilator designed by one of the authors, which supplied room air at a tidal volume of 4 to 6 mL, a respiratory rate of 70 to 100 breaths·min⁻¹, and inspiratory pressure of 15/5 cm H₂O. After a standard left posterolateral thoracotomy, the inferior pulmonary ligament was divided. Retracting the lung anteriorly, the pulmonary artery, pulmonary vein, and left main bronchus were exposed and prepared with microsurgical instruments. These structures were encircled with a 3/0 silk ligature after hilar stripping. Heparin (75 units) was administered through the hemiazygos vein, the pulmonary artery and vein were clamped, and a venotomy was performed on the pulmonary side of the clamp. The pulmonary artery was flushed by gravity from a height of 40 cm with low-potassium dextran (LPD) pulmonary flushing solution at room temperature via a 24-gauge cannula (BOC Ohmeda AB, Helsingborg, Sweden). The composition of LPD solution was: Na⁺, 168 mmol·L⁻¹; K⁺, 4 mmol·L⁻¹; Cl⁻, 103 mmol·L⁻¹; Mg²⁺, 2 mmol·L⁻¹; PO₄ ³⁻, 37 mmol·L⁻¹; dextran, 20 g·L⁻¹; pH, 7.45. The osmolarity was 280 mOsm·L⁻¹. Ventilation was continued during flushing. When the lung turned white and a clear solution drained from the venotomy, pulmonary arterial flushing was stopped. In group A, after separating the ventilation cannula from the mechanical ventilator, the lung was deflated, and the bronchus was clamped. In group B, the lung was inflated to a pressure of 10 cm H₂O and the bronchus was clamped. The ischemic time was 60 minutes in both groups. The control group underwent the surgical procedure without lung ischemia.

Venous, bronchial, and arterial clamps were removed in that order. After 5 minutes of reperfusion, the atrial side of the pulmonary vein was clamped and pulmonary venous blood was withdrawn into a heparinized syringe to measure the pH and pulmonary venous oxygen tension. Pneu-monectomy was performed and pulmonary compliance was measured as described by Homatas and colleagues. ² The specimens were fixed in 10% formalin solution and embedded into 3 paraffin blocks, 4 to 6 slices were stained with hematoxylin and eosin. The histopathological examinations were carried out by one pulmonary pathologist, in a double-blinded fashion.

Comparisons among groups were performed with one-way analysis of variance. The Tukey Krammer multiple comparisons test was used (if the value of q was greater than 3,509 and the p value was less than 0.05). All data were calculated as mean ± standard deviation.

Results

Left pulmonary venous blood oxygen tension was not significantly different (p > 0.05) in comparisons of the 3 groups (Table 1). There was also no statistically significant difference in pH among the 3 groups. Isolated compliance of the left lung was significantly lower in group A compared to group B (p < 0.05) and there was a highly significant difference between group A and the control group (p < 0.00l). The difference in compliance was not significant between groups B and C (Table 1).

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Table 1.

Evaluation of Postischemic Lung Function 5 Minutes After Reperfusion

Group	Pulmonary Venous Oxygen Tension (mm Hg)	pH	Compliance (cc·cm−1 H2O)	p
A	78.5±9.38	7.26±0.17	0.71±0.15	< 0.05 (A versus B)
B	79.1±17.5	7.26±0.12	1.31±0.53
C	81.4±11.28	7.3±0.09	1.82±0.71	< 0.001 (A versus C)

Interstitial edema was noted in both experimental groups. In group A, alveolar edema was detected in 3 rats and perivascular edema was present in all 10 (Table 2). Neither alveolar nor perivascular edema was found in group-B rats (Table 3). Peribronchiolar edema was detected in 8 rats in group A and 2 in group B. Congestion was more severe in group-A rats compared to those in group B (Tables 2 and 3). Intrapulmonary hemorrhage was more prevalent and more severe in group A compared to group B where only 2 rats had mild intrapulmonary hemorrhage (Figure 1). No histological abnormalities were noted in the control group.

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Figure 1.

Mild intrapulmonary hemorrhage in rat no. 17 (hematoxylin and eosin stain, original magnification ×125).

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Table 2.

Histopathological Evaluation of Lungs Preserved in an Atelectatic State (Group A)

Rat No.	Interstitial Edema	Alveolar Edema	Perivascular Edema	Peribronchiolar Edema	Congestion	Intrapulmonary Hemorrhage
1	mild	none	mild	none	moderate	mild
2	mild	none	mild	mild	mild	mild
3	mild	none	mild	none	mild	moderate
4	moderate	none	moderate	mild	mild	mild
5	moderate	moderate	mild	mild	mild	mild
6	mild	mild	mild	mild	mild	mild
7	mild	none	moderate	mild	mild	mild
8	mild	none	mild	mild	mild	mild
9	mild	mild	mild	mild	moderate	severe
10	mild	none	mild	mild	mild	moderate

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Table 3.

Histopathological Evaluation of Lungs Preserved in an Inflated State (Group B)

Rat No.	Interstitial Edema	Alveolar Edema	Perivascular Edema	Peribronchiolar Edema	Congestion	Intrapulmonary Hemorrhage
11	mild	none	none	none	none	none
12	mild	none	none	none	none	none
13	mild	none	none	none	none	none
14	mild	none	none	mild	none	none
15	mild	none	none	none	none	none
16	mild	none	none	none	mild	none
17	mild	none	none	none	mild	mild
18	mild	none	none	none	mild	mild
19	mild	none	none	none	mild	none
20	mild	none	none	mild	mild	none

Discussion

The lung, among all solid organs, is the only one not dependent on perfusion for cellular gas exchange. Cellular ventilation and oxygenation take place on the alveolar surfaces. The pulmonary parenchyma remains viable even after transplantation, although its systemic perfusion is disrupted. ⁵ There are several reports of significant benefits from inflating the lungs during pulmonary preservation but all previous studies were carried out at a follow-up period of at least one hour. The benefits of inflation can be summarized as: prevention of alveolar collapse and micro-atelectasis by continuing surfactant production; prevention of alveolar and peribronchiolar edema; continuation of aerobic metabolism in the pulmonary parenchyma, leading to a decrease in the production of oxygen free radicals; and easy flow of the flushing solution through the whole parenchyma. ^1,6,7

In this study, oxygen tension and pH of the left pulmonary venous blood, pulmonary compliance, and histopa-thological changes in the parenchyma were evaluated as parameters of postischemic injury at 5 minutes after 60 minutes of normothermic ischemia in both atelectatic and hyperinflated lungs. Although there was no statistically significant difference between pulmonary venous oxygen tension and pH in the experimental groups, the values in the hyperinflated group were closer to those of the control group. Pulmonary compliance was considered to be a reliable marker of ischemic injury and preservation of the lung in a number of previous studies. ^{2,8 –12} In this study, there was a significant difference between the pulmonary compliance in the experimental groups, indicating less impairment in the hyperinflated lungs.

Histopathological studies have generally been performed several days postoperatively when the lung may have repaired itself or early injury may have been missed. In this study, the histopathological changes at 5 minutes after reperfusion were evaluated. Perivascular, peri-bronchiolar, alveolar, and interstitial edema, congestion, and inflammation are considered to be signs of mild to moderate injury, whereas, intrapulmonary hemorrhage, serious vascular congestion, and intravascular thrombosis are the signs of severe injury. ^13,14 The histopathological examinations in this study showed that signs of mild to moderate injury were seen more often in the atelectatic lungs compared to the hyperinflated lungs. Signs of severe injury, particularly intrapulmonary hemorrhage, were also more frequent and more severe in the atelectatic group. Intravascular thrombosis, an indicator of severe injury, was not seen in either of the experimental groups. Peribronchiolar, interstitial, and alveolar edema, and congestion are the factors that decrease pulmonary compliance and it is clear that pulmonary compliance and histopathology immediately after reperfusion were better preserved in the hyperinflated group. Significant changes in blood gas values might be expected in the early postreperfusion period due to the decrease in pulmonary compliance and histopathological changes, as mentioned in previous reports. However, neither pulmonary venous oxygen tension nor pH reflected the extent of functional and structural changes at 5 minutes postreperfusion in this study.

It was concluded from these findings that postischemic injury occurred at an early stage in atelectatic lungs, before any change in blood gas values. Superior postischemic preservation was achieved in lungs maintained in a hyperinflated state.