Preliminary evidence that hydrostatic edema may contribute to the formation of diffuse alveolar damage in a Holstein calf model

Background: Two notable findings of clinically healthy feedlot cattle suggest they may have pulmonary hydrostatic edema during the finishing phase of production: increased pulmonary arterial wedge pressures and pulmonary venous hypertrophy. The goal of this study was to determine if increased pulmonary arterial wedge pressure (PAWP) in a Holstein calf could lead to diffuse alveolar damage consistent with the early, exudative phase of acute interstitial pneumonia of feedlot cattle. Methods: Six male Holstein dairy calves were given daily subcutaneous injections of the nonspecific ß-adrenergic agonist isoprenaline (10 mg/kg/d), to induce left ventricular diastolic dysfunction, or sterile water for 14 days. On Day 14, pulmonary arterial pressures and wedge pressures were measured, echocardiography performed, and the ratio of mitral valve flow velocity (E) to septal lengthening velocity (e’) calculated. Calves were euthanized on Day 15 and lung lesions semi-quantitatively scored. Results: Mean PAWP was 12 ± 1 mm Hg in calves that received isoprenaline and 7 ± 1 mm Hg in controls ( P = 0.01). Calves that received isoprenaline tended to have greater relative wall thickness than control calves ( P = 0.15) and greater E/e’ ratios ( P = 0.16), suggestive of concentric hypertrophy and diastolic dysfunction, respectively. Calves that received isoprenaline also tended to have a left ventricle and interventricular septum that was 29 ± 10 g heavier than control calves ( P = 0.10) when controlling for body mass. Hyaline membranes, the hallmark feature of diffuse alveolar damage, were evident in lung sections from all calves that received isoprenaline but none of the controls. Conclusions: Consistent with prior pathological and physiological studies of feedlot cattle, this study provides preliminary evidence that cattle presenting with clinical signs and pathology consistent with early stage acute interstitial pneumonia could be attributable to hydrostatic edema associated with left ventricular failure.


Introduction
Feedlot cattle are susceptible to two diseases of the cardiopulmonary system as they approach slaughter weight: congestive heart failure 1 and acute interstitial pneumonia (AIP) 2 . In human medicine, and for the purpose of our study, acute interstitial pneumonia was defined as an acute respiratory distress syndrome (ARDS) of unknown etiology that is not primarily attributable to hydrostatic edema due to congestive left ventricular failure, pulmonary venous hypertension, or fluid overload 3 . In veterinary medicine, a consensus definition of ARDS has been proposed 4 . Diagnosing feedlot cattle using the proposed criteria, however, is not feasible largely because the diagnostic tools for ruling out hydrostatic edema are not available; consequently, it has been said that definitive diagnosis of AIP in cattle requires histopathologic evaluation of lung tissue obtained postmortem 5 . The histological counterpart of ARDS is typically diffuse alveolar damage (DAD), defined by the presence of hyaline membranes 6 .
If an animal dies in the acute, exudate phase of AIP, then hyaline membranes and hemorrhage may be the only lesions observed 5 . This is problematic because these lesions are not pathognomonic for ARDS and may be attributable to hydrostatic edema. Furthermore, there are two notable findings of clinically healthy feedlot cattle that suggest they may be at risk of congestive left heart failure, and therefore pulmonary hydrostatic edema, during the finishing phase: increased pulmonary arterial wedge pressures (PAWP) and pulmonary venous hypertrophy 7 . This leads us to speculate whether cattle presenting with clinical signs and pathology consistent with early stage AIP could, at least in some instances, be attributable to hydrostatic edema associated with left ventricular failure. The goal of this study was to determine if increased PAWP due to left ventricular dysfunction in a Holstein calf model could lead to pulmonary lesions consistent with the early, exudative phase of AIP in feedlot cattle.

Methods
Overview Six, day-old male, intact clinically healthy Holstein dairy calves were collected from a farm in West Texas and individually housed under normoxic conditions (altitude: 975 m). At 7-days of age (Day 1 of the study), calves (n = 3) were given daily subcutaneous injections of the nonspecific ß-adrenergic agonist isoprenaline (10 mg/kg/d) (henceforth, ISO calves) or an equivalent volume of sterile water (controls, n = 3) for 14 days. On Day 14, pulmonary arterial pressures and wedge pressures were measured and echocardiography performed. Calves were euthanized on Day 15. The heart and lungs were weighed, and lung sections histologically evaluated.

Ethical considerations
Institutional Animal Care and Use Committee approval was granted prior to initiation of the study (Protocol 17013-02). Efforts were made to ameliorate animal suffering by following recommendations for the housing and care of animals provided by the National Research Council 8 .
Study site, housing, and feed Day-old male, intact, male Holstein calves were obtained from one commercial dairy in West Texas. Six calves were included in this preliminary study as, to our knowledge, no similar studies had been performed in this species. Calves were fed 4 to 5 L of colostrum within 12 hours of birth. They were given 3 L of non-medicated milk replacer (22% crude protein, 15% crude fat) twice per day throughout the study (Purina® Herd Maker®, Gray Summit, MO, USA) and had ad libitum access to water. From 1 week of age (Day 1) calves had ad libitum access to a calf starter (Purina®, Ampli-Calf® Starter 22, Gray Summit, MO, USA) with 22% crude protein (dry matter basis).
All calves were individually housed in pens with dimensions 1.8 m by 2.3 m (Agri-Plastics, Grassie, ON, Canada). Four calves were housed on a raised slatted floor inside temperature-controlled chambers (temperature 17 ± 3°C). Calves were housed according to body mass so that the heaviest calves at the start of the study (one control and one experimental calf) were housed in one chamber (Calves 4 and 5) and the lightest calves (Calves 1 and 2) in another chamber. One control (Calf 3) and one experimental calf (Calf 6), were housed in shaded outdoor pens with straw bedding on a sloped concrete floor. The pens were moved to new locations every 3 days and the inside of the pens cleaned with disinfectant (Virkon S, DuPont, Wilmington, DE, USA). Soiled straw was removed once daily, and all straw was replaced every 3 days.

Isoprenaline/Isoproterenol
To our knowledge, isoprenaline (DL-Isoproterenol hydrochloride, 98%, Alfa Aesar, Ward Hill, MA, USA) has not been used in large animal models of heart failure with preserved ejection fraction (HFpEF). The dosage used in this study (10 mg/kg sid) was double the dosage used in a study of equivalent duration in a mouse model 9 . The isoprenaline was dissolved in sterile water (50 mg/mL) at room temperature prior to subcutaneous injection in the neck. Alternating sides of the neck were used. Controls were injected subcutaneously in the neck with an equivalent volume of sterile water (0.2 mL/kg). Treatments were given between 8:30 am and 9:30 am after all calves had been fed milk replacer. Injections were given whilst calves were manually restrained within their pen.
Pulmonary arterial pressure measurement Pulmonary arterial pressure (PAP) testing was performed on Day 14. Calves were restrained in a calf chute and a halter used to hold the calf's head to one side so that the right jugular grove was exposed. The neck was clipped and cleaned with chlorhexidine solution. A 7-French peel-away introducer (IS-07AS, Vascor Medical Corporation, Tarpon Springs, FL, USA) was placed in the jugular vein prior to inserted of a 110 cm, 7 French, polyurethane, modified J-tip wedge pressure catheter (172-110P, Vascor Medical Corporation, Tarpon Springs, FL, USA). A pressure transducer (TranStar DPT, Smiths Medical ASD, Inc., Dublin, OH, USA) was interposed between the catheter to the data acquisition system (IX-TA-220, iWorx Systems, Inc., Dover, NH, USA). The pressure waveforms were recorded and analyzed offline (LabScribe3, iWorx Systems, Inc., Dover, NH, USA). Pressures were analyzed at the end of expiration. The position of the catheter tip was determined by monitoring the change in the pressure waveform as the catheter tip was advanced through the right atrium, right ventricle, and finally into the pulmonary artery.

Echocardiography
Echocardiography was performed on Day 14. Calves remained standing throughout the procedure. Fractional shortening and ejection fraction (cubed or Teichholz method 10 ) were obtained from right parasternal long-axis views of the left ventricle obtained between intercostal spaces 3 to 6. Relative wall thickness was calculated as two-times the left ventricular free wall diastolic thickness divided by the left ventricular internal diastolic diameter. Early diastolic (e') septal lengthening velocities and early mitral flow (E) velocities were obtained from a left parasternal apical four-chamber view. The E/e' ratio is a measure of left ventricular filling pressure and, consequently, a diagnostic measure of diastolic dysfunction 11 .

Postmortem examination and histology
Calves were euthanized with intravenous pentobarbital sodium (85 mg/kg) on Day 15 of the study and exsanguinated. Lung lobes were individually weighed. The atria were separated from the ventricles at the atrioventricular junction. The right ventricular free wall (RV) was separated from the left ventricle and septum (LVS). The RV and LVS were individually weighed.
The left diaphragmatic lobe was perfused with formalin (10%, neutral buffered) at 15 to 20 cm H 2 O for approximately 5 minutes. After 5 days of formalin fixation, lung sections were collected midway along the dorsal aspect of the lobe for histology. Paraffinized sections (4 µm) were stained with hematoxylin and eosin and Masson's trichrome. Lung sections were semi-quantitatively scored based on severity (0 = no lesion; 1 = mild; 2 = moderate; 3 = severe). The investigator was blinded to the calves' identities and treatment group. The pulmonary parenchymal lesions scored included interstitial edema, intra-alveolar edema, hemorrhage and hemosiderin, inflammatory infiltrates, type II hyperplasia, and interstitial fibrosis. Pulmonary arterioles (< 500 µm) and veins were scored for medial hypertrophy and adventitial fibrosis. Veins were distinguishable from arteries as they have spiral bundles of muscle that give them a beaded appearance.

Statistical analyses
Statistical analyses were performed using commercial software (Stata version 12.1, College Station, TX). Calf 1 was excluded from statistical analyses because a sub-endocardial lesion was detected postmortem that likely affected his cardiac function and, consequently, pulmonary vascular pathophysiology. Between group differences were evaluated using Student's t-test with equal variances. Student's t-test is a suitable statistical method for small sample sizes (n ≤ 5) even if group sizes are unequal as long as the effect size is expected to be large. 12 Linear regression was used to determine if there was a significant difference between groups in the mass of the right ventricle, left ventricle and interventricular septum, and lung, while controlling for body mass.

Cardiopulmonary pressures
Calves that received ISO had a greater mean PAWP but a lower mean PAP than controls. Mean PAWP was 12 ± 1 mm Hg in the ISO group and 7 ± 1 mm Hg in the control group (P = 0.01). Mean PAP was 23 ± 2 mm Hg in the ISO group and 43.5 ± 8 mm Hg in the control group (P = 0.05). The control group had a greater heart rate (104 ± 9 bpm) than the ISO group (74 ± 4 bpm; P = 0.03). Results are presented in Table 1.

Echocardiography
There was no statistical difference in ejection fraction or fractional shortening between the ISO and control groups indicating that left ventricular systolic function was preserved. Ejection fraction was 66 ± 6% in the control calves and 54 ± 6% in the ISO group (P = 0.29). Fractional shortening was 36 ± 4% in the control calves and 29 ± 4% in the ISO group (P = 0.27). ISO calves tended to have greater relative wall thickness than control calves (P = 0.15) and greater E/e' ratios (P = 0.16) which is suggestive of concentric hypertrophy and diastolic dysfunction, respectively. Results are presented in Table 2.

Pathology
There was no difference in body mass at the end of the study (P = 0.51). The mean body mass was 46.6 kg ± 3.3 kg for the control calves and 42.8 ± 3.4 kg for the ISO group. Calf 1 had endocardial fibrosis extending from the atrio-ventricular boundary to the dorsal aspect of the papillary muscle and approximately 0.5 cm into the myocardium (Supplementary file). Calf 5 had a 1 cm abscess in the right diaphragmatic lobe and Calf 6 had acute fibrinous pneumonia affecting the ventral 20% of the right cranio-ventral lung lobe (Supplementary file). The lungs of Calf 2  The ISO group tended to have a left ventricle and interventricular septum that was 29 ± 10 g heavier than control calves (P = 0.10) when controlling for body mass. There was no difference between groups in the mass of the lung (P = 0.81) or right ventricle (P = 0.36) when controlling for body mass. Results are presented in Table 3.
In general, tissue areas with lesions consistent with DAD were multifocal and often located adjacent to a lobule with a normal healthy appearance. Occasionally, a gradual change from healthy to DAD was observed within the same lobule. All ISO calves had mild or moderate interstitial and intra-alveolar edema, mild or moderate hemorrhage, and type II hyperplasia. Two control calves (Calves 3 and 5) showed mild interstitial edema in some areas. Interstitial inflammatory infiltrates were evident in two calves, particularly Calf 1. No calves showed evidence of interstitial fibrosis. Calves 4 and 6 showed moderate epithelisation of alveoli. Calf 6 also had severe emphysema and honeycomb parenchymal remodeling (Figure 1). Results are presented in Table 4.
Mild or moderate medial hypertrophy and adventitial fibrosis of pulmonary arterioles was evident in all calves. ISO calves had more substantial medial hypertrophy of the pulmonary veins than controls. Pulmonary veins were not evident in Calf 6. Adventitial fibrosis was not observed except in Calf 4. Results are presented in Table 5.    Table 5. Semi-quantitative assessment of medial hypertrophy and adventitial fibrosis in pulmonary arteries (< 500 µm) and veins from male intact Holstein calves after 14 days of receiving the nonspecific ß-adrenergic agonist (ß-AA) isoprenaline (10 mg/kg/d) and control calves that received an equivalent volume of sterile water (0.2 mL/kg).

Discussion
The findings of this study indicate that left ventricular dysfunction could feasibly contribute to the development of diffuse alveolar damage, the pathologic correlate of acute interstitial pneumonia, an ARDS of feedlot cattle of unknown etiology. Two weeks of daily injections with isoprenaline led to the development of heart failure with preserved ejection fraction (HFpEF); left ventricular systolic function was preserved but diastolic function was reduced. Given that subclinical HFpEF in a Holstein model has many of the pathologic features and physiologic measurements consistent with ARDS 13 , this preliminary study indicates that the diagnosis of AIP in a feedlot animal can only be considered as tentative if left ventricular dysfunction has not been ruled out as a primary cause.
The PAWP of the ISO calves were significantly greater than controls but substantially less than the PAWP of 26 mm Hg previously reported in feedlot cattle at the end of the feeding period at the altitude of 1,220 m 7 . Pulmonary arterial wedge pressures are typically less than 15 mm Hg in non-feedlot cattle and other mammals 14 ; therefore, the DAD seen in the ISO calves in this study is likely attributable to the acute doubling of PAWP over a 2-week period. Feedlot cattle may be able to tolerate much greater PAWP if pressures were to rise insidiously throughout the feeding period. There are a variety of adaptations, in addition to pulmonary arterial and venous remodeling 15 , that may occur in response to rising pulmonary vascular pressures: first, the lymphatic system is recruitable -it is able to increase the rate of lung-water clearance by over 10-fold. 16,17 ; and second, alveolar fibrosis may occur to reduce alveolar-capillary membrane permeability to water 18 . The time period over which pulmonary vascular pressure rise is, therefore, likely to be a key determinant of the pulmonary adaptability.
It is also feasible that isoprenaline induced DAD through forward hemodynamic effects. Increased cardiac contractility and pulse pressure may have had deleterious downstream effects on the pulmonary vasculature 19,20 paving the way for increased fluid flux into the pulmonary interstitium. Whether attributable to backward or forward hemodynamic effects, an increase in pulmonary capillary pressure will lead to mechanical injury of the alveolarcapillary membrane leading to increased capillary permeability and impaired gas exchange 21 . Although acute damage can be reversed, long term blood-gas barrier disruption leads to lung fibrosis, inflammation, impaired alveolar fluid clearance, and muscularization of pulmonary vessels 21,22 .
Interestingly, the two calves housed in shaded outdoor pens on straw bedding had the greatest pulmonary arterial pressures within their respective treatment groups. In a prior study, calves housed on straw that developed bloody scours had histologic evidence of DAD and greater pulmonary arterial pressures than calves housed on slatted flooring that did not develop scours 23 . It was speculated that intestinal disease may contribute to the development of pulmonary disease in cattle 23 . Calves housed on straw bedding in our study may have been in the incubation phase of intestinal disease.
Even though there were only 6 calves in this study, the goal of the study, to determine if left ventricular dysfunction could lead to lesions consistent with the early, exudative phase of AIP, was met. For ethical reasons, decompensated heart failure was not induced; therefore, it is not possible to say whether overt left ventricular failure clinically manifests as an ARDS-like event. Human medical reports suggest, however, that it can present as an ARDS which is why the diagnosis of ARDS requires that acute dyspnea attributable to hydrostatic edema must first be ruled out 3 . Large-scale prospective cohort studies of feedlot cattle are necessary to determine if pulmonary arterial pressures and PAWP are positively associated with risk of respiratory diseases, such as AIP. We speculate that elevated pulmonary vascular hydrostatic pressures may act synergistically with mediators of acute alveolar damage, such as pathogens or airborne irritants, to promote the development and progression of respiratory disease.

Data availability
All raw data underlying this study is available from Harvard Dataverse under a CC0 Public Domain Dedication

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work. In this small pilot study authors tried to determine if increased pulmonary arterial wedge pressure (PAWP) in a Holstein calf could lead to diffuse alveolar damage (DAD), which provide histologic evidence to diagnose acute interstitial pneumonia (AIP). The results showed that left ventricular dysfunction can associate with, or be one of the cause that a feedlot steer has the sign of AIP (hyaline membranes and type II pneumocyte hyperplasia).
"If applicable, is the statistical analysis and its interpretation appropriate?" Due to the small number of animals used in the study, a qualified statistician is suggested to verify the statistical analyses.
"Are the conclusions drawn adequately supported by the results?" More histology images (figure 1) with clear labels of different type of lesions and pathology findings will help to support the findings shown in Table 4 and 5.
Specific comments:

Methods Postmortem exam and histology
Whether a board certified veterinary pathologist identified and scored the histologic lesions?

Results
More histology images (Figure 1) with clear labels of different type of lesions and pathology findings will help to support the findings shown in Table 4 and 5.
2. In the introduction or discussion part, I would like authors to refine the novelty of this study. Increased PAWP due to left ventricular dysfunction can lead to hydrostatic edema, diffuse alveolar damage, all of which are pathologically correlate with acute interstitial pneumonia. This concept is acknowledged in the which are pathologically correlate with acute interstitial pneumonia. This concept is acknowledged in the cattle field and the results from this study confirmed this concept and suggest that in feedlot, ARDS can not be diagnosed without first rule out LV dysfunction.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. For a small pilot study, the approach to statistical analysis is acceptable. However, in some cases the statistical analysis may have provided results that are misleading. For example: in the Results section, the authors state that there was no difference in body mass between calves in the two groups. How was this tested? If by the Student's t-test: per the Methods section, the t-test is only appropriate for small samples of unequal size if the effect is expected to be large. But the difference in body mass between the groups was not large, so it's not convincing that the results of this analysis are accurate. Body mass of the calves is not a particularly important outcome of this study, but if differences are going to be reported, a different statistical test should be used.
Similarly, while the ejection fraction between the two groups was not significantly different, the distribution of values for this outcome in the calves in the two groups looks like EF would likely have been significantly Similarly, while the ejection fraction between the two groups was not significantly different, the distribution of values for this outcome in the calves in the two groups looks like EF would likely have been significantly lower in treated calves if more animals had been included. That is, the study likely lacked power to identify a true difference in ejection fraction. Thus this model may not actually induce left heart failure with preserved ejection fraction, as the authors contend.
"Are the conclusions drawn adequately supported by the results?" Generally yes, with the caveat that the conclusions are based on a very small number of animals. The data depicted in Table 4 support the authors' contention that experimentally-induced left heart dysfunction was associated with lung lesions typical of early acute interstitial pneumonia: hyaline membranes and type II pneumocyte hyperplasia.
Specific comments:

Postmortem exam and histology
What were the qualifications of the investigator who identified and scored the histologic lesions? Was that person a board certified veterinary pathologist?

Results
Do the investigators have any idea why the ISO treated calves had lower heart rates than the control calves?

Discussion
The writers note: "…this preliminary study indicates that the diagnosis of AIP in a feedlot animal can only be considered as tentative if left ventricular dysfunction has not been ruled out..." Strictly speaking, AIP (acute interstitial pneumonia) is a pathologic diagnosis, which can result from a variety of possible primary insults. This study does suggest that acute left heart dysfunction can be associated with the lung lesion consistent with early AIP (hyaline membranes and type II pneumocyte hyperplasia). A histologic diagnosis of AIP can be made based on evidence of diffuse alveolar damage; this work suggests that any cause of acute left heart failure could be one of several possible causes of AIP. This is in contrast to the clinical definition of ARDS which, as the authors note in the introduction, currently requires rule out of respiratory failure solely due to heart failure. However, it is usually not possible in the feedlot to make the measurements necessary to confirm ARDS and rule out acute left heart failure, based on current definitions. This research does support the concept that a feedlot steer or heifer with acute severe dyspnea may be suffering from a lung lesion resulting solely from acute congestive left heart failure, as well as a lung lesion resulting from infection, pneumotoxicity, or other possible causes of AIP.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly No competing interests were disclosed. Competing Interests: Referee Expertise: Lung disease of cattle, including lung infection, and immunity to lung disease caused by viruses or bacteria I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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