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The bronchial circulation plays a significant role in the pathophysiological changes of burn and smoke inhalation injury. There is a marked increase in bronchial blood flow immediately after inhalation injury. In this study we examine the ablation of bronchial artery will attenuate the pathophysiological changes and improve survival rate after burn and smoke inhalation injury in ovine model. Acute lung injury was induced by 40% total body surface area 3rd degree cutaneous burn and cotton smoke (48 breaths of cotton smoke, <40°C) under deep anesthesia. Twelve adult female sheep were divided into two groups: 1) Sham (injured, not ablated bronchial artery, n=6); 2) Ablation (injured, ablated bronchial artery, n=6). Ablation of bronchial artery was performed 72h before-injury. The experiment was continued for 96h. Burn and smoke inhalation injury significantly increased the regional blood flow in Bronchi. Ablation of bronchial artery significantly reduced the acute regional blood flow increase in proximal and distal bronchi. All animals in the ablation group survived until 96h. Four of them were successfully weaned from the ventilator. Three of sham group animals met euthanasia criteria at 60h and one of them met the criteria at 78h. The lung wet/dry weight ratio, the histology score, and myeloperoxidase activity were significantly increased by the insult, but ablation of bronchial artery attenuated these changes. Burn and smoke inhalation injury induced a significant increase in bronchial blood flow and accelerated airway obstruction, pulmonary vascular changes, pulmonary edema and pulmonary dysfunction. The ablated bronchial circulation attenuated these pathophysiological changes.
Burn injury is very traumatic, especially when the thermal injury is associated with smoke inhalation. This combination greatly increases morbidity and mortality [1, 2]. After smoke inhalation, there is a rapid appearance of hyperemia in the upper airway of humans and sheep [3–5]. Hyperemia of the airway is the most effective way of diagnosing smoke inhalation . Pulmonary edema has been directly related to smoke inhalation injury as evidenced by an increase in extravascular lung water and lung lymph flow after inhalation injury [7–11]. Smoke inhalation has also been shown to cause an increase in microvascular permeability to protein in the pulmonary and bronchial circulations [8–10, 12]. These physiological alterations in pulmonary microvasculature are delayed in onset, and the peak of increased microvascular permeability was observed around 24h after injury . In contrast, there is a marked increase in bronchial blood flow immediately after inhalation injury [3, 14]. Because the increased bronchial blood flow largely enters the pulmonary vasculature through pre-capillary anastomoses with the pulmonary microcirculation [15–17], it has been suggested that the bronchial circulation plays a significant role in the spread of injury from the airway of the lung to the parenchyma [11, 18]. Reduction of the bronchial artery reduced lung edema formation after inhalation in anesthetized canine model  and in a conscious sheep model [10, 18]. We performed 96hr survival experiments and analyzed the regional tissue blood flow. The present study was undertaken to provide a detailed analysis of pulmonary function at 96hr after injury and the 96hr survival rate. We hypothesized that ablation of the bronchial artery would not only reduce pulmonary injury following combination burn and inhalation injury but also allow survival to 96hr with weaning from ventilatory support.
Twelve adult female sheep (30–40 kg) were cared for in the Investigative Intensive Care Unit at our institution. The experimental procedure was approved by the Animal Care and Use Committee of the University of Texas Medical Branch. The National Institutes of Health and American Physiological Society guidelines for animal care were strictly followed. The Investigative Intensive care unit is accredited by The Association for the Assessment and Accreditation of Laboratory Animal Care International.
All animals were endotracheally intubated and ventilated during the operative procedure while under isoflourane anesthesia. Arterial catheters (16 gauge, 24 in., Intracath, Becton Dickinson, Sandy, UT) were placed in the descending aorta via the femoral artery. A Swan-Ganz thermal dilution catheter (model 93A-131-7F, Edwards Critical-Care Division, Irvine, CA) was positioned in the right pulmonary artery via the right external jugular vein. A silastic catheter was positioned in the left atrium through a left thoracotomy. A 10-mm pneumatic occluding cuff (In Vico Metrics, Healdsburg, CA) was implanted around the left pulmonary artery just distal to its origin. Sheep have a single bronchial artery, which was called “carinal artery” . In the ablation group (n=6), the bronchial artery “carinal artery” was exposed near its origin on the aorta through a left 4th thoracotomy, and 1mL of 70% ethanol were injected into the artery after the ligation of the bronchial artery with 5-0 silk suture. In sham group (n=6), the bronchial artery was exposed but left intact.
After a recovery period, the sheep were deeply anesthetized with isoflourane and were given a flame burn [40% total body surface area (TBSA), third degree] and inhalation injury (48 breaths of cotton smoke, <40°C). After burn and inhalation injury, all sheep were placed on a ventilator with positive end-expiratory pressure set to 5 cm H2O and tidal volume maintained at 15 mL/kg in an awake condition. The latter tidal volume is equal to about 10 mL/kg in humans due to the large ventilatory dead space of sheep . All animals were given fluid resuscitation with Ringer’s lactate solution strictly according to the Parkland formula (4 mL/kg/% TBSA burned/24 hr) . The experiment was continued for 96 h. The ventilation weaning was begun if the animal was clinically stable and her PaO2/FIO2 was higher than 250 at 48h after initial insult. In ventilation weaning, the ventilation setting was changed to pressure support ventilation (PSV) in combination with synchronized intermittent mandatory ventilation (SIMV) and tried SIMV with a low rate and low-level PSV, then transitioned to continuous positive airway pressure (CPAP). If the animals’ condition remained steady, the animal was taken from ventilatory support and transferred to blow-by. Escharectomy of cutaneous wound was not performed but the wound was disinfected with Betadine Solution (Purdue Pharma, Stamford, CT) every day. Euthanasia criteria were determined when systolic blood pressure was below 60 mmHg, heart rate continued below 40/min for 1 hour, PaCO2 was above 100 Torr, PaO2 was below 45 Torr (FIO2 at 100%). If the animal met one of these criteria, the measurement was repeated after another 1 hour. If the animal met the criteria again, it was anesthetized with ketamine and euthanized by high dose of i.v. KCl (60ml).
To substantiate the changes in bronchial blood flow, flow was measured with the fluorescent microsphere technique (Interactive Medical Technologies, West Los Angeles, CA) which we had previously developed . After operation, before injury and 3, 6, 24, 72, 96 h after injury, ~12 × 106 fluorescent colored microspheres (15.0 ± 0.1 μm) were injected into the left atrium. Since some of the microspheres may pass through some of the larger vessels in the non bronchial pulmonary circulation, the pneumatic occluder on the left pulmonary artery was inflated immediately after microsphere injection to prevent any pulmonary artifact to the bronchial blood flow measurement . To calibrate microsphere numbers per blood flow, blood was withdrawn from the aorta with a Harvard pump (Harvard Apparatus model 55-1143, South Natick, MA) at a rate of 10 mL/min; the withdrawal was started before microsphere injection and continued for 2 min. Trachea, left proximal bronchi (6–8 mm bronchi near carina) and left distal bronchi (2–4 mm bronchi in distal lower lobe) were obtained postmortem and used to quantify the bronchial blood flow to the bronchi. The parenchymal tissues were removed from the latter by careful dissection.
Arterial and mixed venous blood samples were taken at different time points for measurement of blood gases (IL GEM Premier 3000 Blood Gas Analyzer; GMI, Minnesota) and white blood cells, neutrophil counts were also counted at different time points (HEMAVET® HV950FS; Drew Scientific, Inc, Texas). PaO2/FIO2 ratio was measured to help assess pulmonary gas exchange. Pulmonary shunt fraction (Qs/Qt) was calculated using standard equations. Sheep were sacrificed under deep ketamine anesthesia 96 h after injury or at the time sheep met euthanasia criteria.
For histological assessment of the lung tissue, the right lung was then removed, and a 1-cm-thick section was taken from the middle of the lower lobe, gently injected with 10% formalin in various areas to facilitate fixation and immersed in formalin and fixed for 2 to 3 days. The slice of tissue was then sampled per protocol by a technician unaware of the study conditions into four samples representing approximately 60% of the initially collected lung slice. Fixed samples were then embedded in paraffin, sectioned at 4μm, and stained with hematoxylin and eosin. A pathologist without knowledge of the group assignments evaluated the lung histology. Levels of airway obstruction were obtained with a standardized protocol by estimating the degree of obstructive material (0 to 100%) localized within the airway lumen. Typically between 10 and 20 bronchi and 100 to 200 bronchioles are scored per animal. Mean bronchial and bronchiolar obstruction were obtained from these estimated [23, 24]. The parenchyma from each of the four sections from each animal were also examined and semiquantitatively scored for the degree of alveolar edema and the presence of neutrophils localized in the alveolar space. A score of 0 was used to represent the absence or normal appearance of the parenchyma, with scores of 1–4 representing increasing areas of the histological section exhibiting the pathological parameter. A mean score for each parameter was calculated from the four individual scores. The remaining lower one-half of the right lower lobe was used for the determination of bloodless wet-to-dry weight ratio .
The activity of myeloperoxidase (MPO), an indicator of neutrophil accumulation, was determined spectrophotometrically in whole lung homogenates. MPO concentrations were evaluated on homogenized right lung with a commercially available assay. Myeloperoxidase activity was reported as U/g tissue (CytoStore, Calgary, AB, Canada)
Summary statistics of data are expressed as means ± standard error of the mean. Statistical significance was determined using an analysis of variance, and for the differences between each value, Tukey-Kramer honestly significant difference was used (post-hoc analysis). Effects and interactions were assessed at p < 0.05 level of significance. Chi-square analysis of survival differences was calculated between ablation and sham groups.
Figure 1 shows the regional blood flow changes measured with fluorescent colored microspheres. In the trachea, burn and smoke inhalation injury significantly increased in both injection and sham groups (Ablation group; Base Line:0.18±0.04 vs. 6h:1.25±0.22, Sham; Base Line:0.15±0.02 vs. 6h:1.67±0.16 mL/g tissue). The regional blood flow at 6h in trachea had 6.9 and 11 times higher than base line and the ablation of the bronchial artery didn’t reduce the acute regional blood flow increase in trachea (Figure 1A). In the proximal and distal bronchi, burn and smoke inhalation injury significantly increased the regional blood flow at 6h, 10 times and 9.6 times higher than base line in sham group (Proximal bronchi; Base Line:0.24±0.01 vs. 6h:2.47±0.18, Distal Bronchi; Base Line:0.30±0.04 vs. 6h:2.89±0.42). Ligation and ethanol injection in the bronchial artery significantly reduced the acute regional blood flow increase in proximal and distal bronchi (Proximal bronchi; Base Line:0.13±0.02 vs. 6h:0.51±0.25, Distal Bronchi; Base Line:0.20±0.04 vs. 6h:0.44±0.25). Notably, smoke inhalation did not induce hyperemic changes in proximal and distal bronchi in the ablation group. However, the regional blood flow in ablation group was slightly increased between after injection and base line (after 72h) (Proximal bronchi; After Injection:0.08±0.02 vs. Base Line:0.13±0.02, Distal Bronchi; After Injection:0.15±0.05 vs. Base Line:0.20±0.04) (Figure 1B,C).
Figure 2 shows the animal survival proportions. All animals in ablation group survived until 96h. Four of them were successfully weaned from the ventilator but two of them didn’t meet the ventilation weaning criteria (PaO2/FIO2 >200 at 48h). None of the animals in sham group met criteria for the removal from the ventilator. Three of these sheep in the sham group met euthanasia criteria at 60h and one of them met the criteria at 78h. There is a significant difference between ablation group and sham group (p=0.02).
Figure 3 shows the effect of ablated bronchial artery blood flow on pulmonary gas exchange. In PaO2/FIO2 ratio graph, four of injection animals did not fall below 200 within 48h but two of them dropped below 200. All sham animals dropped below 200 within 48h. After 48h, four of injection animals were started on ventilation weaning because they met the ventilation weaning criteria. Two of them didn’t meet criteria, so they were not started ventilation weaning. None of the animals in the sham group met the criteria. The graph lines were separated after 48h in the ablation group because the ventilation settings were different. There was no significant difference but overall the ablation group had higher PaO2/FIO2 ratios than the sham group. In the pulmonary shunt fraction graph, there was no significant difference but overall, the ablation group had a lower pulmonary shunt fraction than sham group.
Figure 4 shows the effect of ablated bronchial artery blood flow on pulmonary circulation. Overall, the sham group had higher pulmonary artery pressure than the ablation group and remained higher until 72h. Significant differences at 12h, 36h and 48h were found between the ablation group and sham group (Figure 4A). Figure 4B shows the pulmonary vascular resistance index. The value was remarkably increased at 12h and remained high until 36h and then decreased in the sham group. On the other hand, the values were lower in the ablation group. Significant differences were found at 12h, 18h, 24h, 30h and 36h.
Figure 5 shows the effect of ablated bronchial artery blood flow on white blood cells (WBC) counts and neutrophil counts. Burn and smoke inhalation injury increased WBC and neutrophil counts in the sham group animals. However, ablation of the bronchial artery attenuated the changes. Significant differences occurred at 24h in neutrophil counts.
We separated the pathological data, wet to dry ratio data and myeloperoxidase data in each survival time hour because these results are different in each time course . In the bronchial artery ablation and no injury animals (control group, n=2), the average airway obstruction score in bronchi and bronchiole were 2.6 and 2.3 respectively. The airway obstruction scores revealed an increase in the mean degree of obstruction in both bronchi and bronchioles in the 60h and 78h sham group. The obstruction score was much lower in the sheep with ablated bronchial blood flow (4.4 ± 1.6). There was a significant difference between the 60h sham group and ablation group (Figure 6A,B). The average scores representing the degree of PMNs and edema seen in the alveolar areas of the lung were 0.6 ± 0.3 and 0.79 ± 0.48, respectively. In the bronchial artery ablation and no injury animals (control group, n=2), the scores representing the presence of PMNs and edema in the alveoli were 0.0 and 0.0 respectively. The PMN cells score and edema formation scores were remarkably increased in sham group. The obstruction score was much lower in the sheep with ablated bronchial blood flow. There was a significant difference between the 60h sham group and ablation group (Figure 6C,D).
In the bronchial artery ablation and no injury animals (control group, n=2), the average lung bloodless wet to dry ratio was 2.9 and 4.4 respectively. Lung bloodless wet to dry weight ratio, a measure of lung water content, was remarkably increased in the sham group. However, the lung wet to dry ratio was much lower in the in the group with ablated bronchial blood flow (4.6 ± 0.5). The activity of myeloperoxidase (MPO), an indicator of neutrophil accumulation, was also remarkably increased in the sham group. However, the MPO activity was much lower in the in the group with ablated bronchial blood flow (Figure 7).
The systemic circulation of the lung supplies nutrient materials to airways, blood vessels, and supporting structures. The bronchial circulatory system has been thought to play a significant role in certain physiological functions such as warming and humidification of inspired air . Normally the bronchial circulation comprises ~1–3% of cardiac output [19, 22]. In several disease states, including pulmonary artery obstruction, congenital pulmonary atresia, chronic bronchietasis, and chronic lung abscess, its proportion of the cardiac output may be much larger [27–29]. Acute elevations of bronchial blood flow have also been noted after infusion of bradykinin, histamine, and endotoxin in dogs and sheep [30–32]. These increases in bronchial blood flow may contribute to airway edema, bronchiectasis, and airway occlusion.
After smoke inhalation, there is a rapid appearance of hyperemia in the upper airway of humans and sheep [3–5]. There is a more than 10 fold increases in bronchial blood flow immediately after inhalation injury [3, 14, 33]. In the present experiment, there is an approximate 10-fold increase at 6h in trachea, proximal and distal bronchi. On the other hand, ligation and sclerosis of the bronchial artery significantly reduced the regional blood flow changes in proximal and distal bronchi. Trachea receives systemic blood supplied from the tracheal bronchial and thoracic tracheal branch of the aorta in sheep . These branches were not ablated in present study because the bronchial artery is the major source of the circulation to the airway, at the level of the carina and below it [19, 34]. Considering the fact, it is easy to understand that the regional blood flow in trachea wasn’t reduced in ablation group and our results also show that these tracheal branches contributed less to lung parenchyma than the bronchial artery itself. The regional blood flow in proximal and distal bronchi slightly increased at base line compared to after ablation. The ablation of bronchial artery was performed 72h before injury. Baile et al.  used radioactive microspheres and reported that ethanol injection (total 4ml) into the bronchoesophageal artery reduced systemic arterial blood flow into the sheep lung by ~75%, whereas ligation of the bronchoesophageal artery reduced by ~50%. We also previously reported that the reduction of airway blood flow in the ligation and 70% 4ml ethanol ablation groups was 32.1±12.0 and 86.3±7.2%, respectively (35). Trachea and bronchi blood circulation have longitudinal anastomosis [36, 37] and only mechanical obstruction of bronchial artery easily leads to opening of the collateral blood flow. In order to ablate the bronchial artery, we ligated with 5-0 silk and injected 1ml ethanol into the bronchial artery but the regional blood flow slightly increased 72h after injection. It was suspected that this increase was because of the collateral blood flow opening and 1ml ethanol was not enough to ablate all anastomoses, some blood flow come back via collateral vessels.
The changes of pulmonary vascular pressure and pulmonary vascular resistance were much greater in out present study of combined burn and smoke injury in comparison to our previous report in which sheep had bronchial artery ablation and the animals had smoke inhalation injury alone (37, 40). Burn and smoke inhalation injury increases reactive oxygen and nitrogen species than smoke inhalation injury alone and these differences might affect the results (32, 42). If only smoke inhalation injury were given, the mortality rate is lower and the changes in WBC, neutrophils, MPO are not as great.
All animals in ablation group survived until 96h and the four of them were succeeded ventilation weaning. Two of them didn’t meet the ventilation weaning criteria at 48h. Whereas, four of sham animals died within 96h and none of these animals met the ventilation weaning criteria at 48h. To our knowledge there is no previous report on the effect of ablation of the bronchial artery on survival, and the success of ventilation weaning in sheep after burn and smoke inhalation injury. The ablated bronchial arterial flow resulted in improved pulmonary gas exchange and pulmonary vascular resistance changes after burn and smoke inhalation injury (Figure 3,4). The mechanism by which the bronchial circulation contributes to the pathogenesis in lung parenchyma has not been clearly defined. With inhalation of smoke, there is a reduction in reflection coefficient (permeability to protein), an increase in filtration coefficient (permeability to small particles), and an increase in pulmonary microvascular pressure [13, 38]. These changes could be virtually eliminated by ablation of bronchial circulation to the lung [10, 11]. The increased bronchovascular permeability accelerates airway exudation and causes airway obstruction. Certainly airway obstruction must also play a role in gas exchange. We have reported a good correlation between airway obstruction and reduced PaO2/FIO2 . However, ablated bronchial circulation attenuated the formation of airway obstruction and improved pulmonary gas exchange.
In the present study, the edema formation score in parenchyma and lung wet to dry ratio were remarkably increased after burn and smoke inhalation injury in sham group but the ablated bronchial artery significantly attenuated the edema formation score in parenchyma and lung wet to dry ratio (Figure 6D, ,7A).7A). Bronchial circulation supplies 88% of the intrapulmonary airway blood flow except the right apical lobe [19, 39] and most of intrapulmonary airway blood flow drained into the pulmonary microcirculation at the pre-capillary level [16, 40], only 13% of bronchial circulation drain into the bronchial venous system into the right heart . Increased bronchial blood flow elevates pulmonary transvascular fluid flux and causes pulmonary edema [38, 42]. Perhaps the answer to the key role of the bronchial circulation is that the venous drainage of the bronchial circulation to the airways that lie within the lung drains into the pulmonary microvasculature while the systemic venous drainage from airways outside the lung drain into the azygos [16, 43]. We confirmed these studies in experiments in which we isolated the bronchial venous drainage to the pulmonary microvasculature .
Neutrophils also play a critical role in burn and smoke inhalation injury induced adult respiratory distress syndrome (ARDS) [45, 46]. In present study, PMN cells score in parenchyma and myeloperoxidase in lung tissue were remarkably increased after burn and smoke inhalation injury in sham group but ablated bronchial artery significantly attenuated PMN cells score in parenchyma and myeloperoxidase in lung tissue (Figure 6C, ,7B).7B). Thus with smoke inhalation injury the airway could release mediators into the bronchial venous drainage that could in turn deliver them to the pulmonary parenchyma where they could elicit lung damage. Under many circumstances one would consider the venous drainage to be somewhat insignificant since it is only 1 % of the total pulmonary blood flow. However in the situations when the blood flow in the bronchial vessel is increased 10 fold it assumes 10% of the total pulmonary blood flow. Neutrophils activated in the bronchial circulation flow out into the pulmonary microcirculation . Treatment of the cells with an antibody to L-selectin prevents the changes in transvascular fluid flux and other aspects of parenchymal damage . Burn and smoke inhalation injury increases bronchial circulation flow and drain out inflammatory mediators and cells into pulmonary parenchyma, whereas ablated bronchial artery reduces a rapid increase of bronchial circulation and suppresses the inflammation. The parenchymal damage which causes pulmonary failure and mortality would be ameliorated by the reduction of rapid bronchial blood flow increase.
In the limitations of this experiment, it is impossible for us to have a true sham group with intact bronchial artery, weaned from the ventilator by 96h. If we compare our survivor animals to the two animals still on the ventilator at 96h and to other injured animals that expired at earlier time periods, there were certainly fewer markers of lung injury in the bronchial artery ligation group. While some might attribute some of this injury to barotraumas from the ventilator, comparison to animals that did not have burn/smoke insult but the same ventilators settings, showed little or no pathological changes.
We succeeded in cannulating a catheter to the bronchial artery while maintaining bronchial artery flow, and injected ethanol 1 hr after injury into the catheter. The ablation of bronchial artery after inhalation injury also attenuated the pulmonary dysfunction induced by burn and smoke inhalation injury. Nonetheless, interventional therapy has progressed rapidly and multiple cannulation techniques have been developed. In the future, we will be able to infuse pharmacological agents into human bronchial arteries. Consequently, it will be possible to suppress the bronchial artery flow in situations of pulmonary hyperemia in humans via the interventional technique.
In conclusion, burn and smoke inhalation injury induced a rapid increase of bronchial blood flow and accelerated airway obstruction, pulmonary vascular changes, pulmonary edema and pulmonary dysfunction. Ablation of the bronchial circulation suppressed the inflammation, attenuated these pathophysiological changes of smoke/burn injury and improved animal survivability. Interventional therapy to modulate the bronchial circulation using pharmacological agents may be an effective strategy in the management of ARDS induced by burn and smoke inhalation injury.
We thank Jeffrey D. Meserve for his editorial assistance and Nettie Biondo and John R. Salsbury for their technical assistance.
This work was supported by National Institute for General Medical Sciences Grant GM66312, GM060688 and Grants 8954, 8450 and 8460 from the Shriners of North America.
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