Lung ultrasound is a reliable method for evaluating extravascular lung water volume in rodents

Thirty male Sprague–Dawley rats (aged 48 weeks, weighing between 225 g and 275 g; Animal Experiment Center, Southern Medical University) were obtained for this study. The study was performed in accordance with the approval and guidelines from the Institutional Animal Care and Use Committee of Guangdong Provincial Hospital of Chinese Medicine, Guangzhou University of Chinese Medicine. All rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg). A tracheotomy was performed, and a tracheal tube was inserted to a depth of 2 cm and secured in place to allow for mechanical ventilation using a rodent respirator (Harvard Apparatus, South Natick, MA). Rats were exposed to fractional inspired oxygen (FiO₂) of 0.21 (room air), respiratory rate 73/min, tidal volume(TV) 4.5 ml/kg and 2 cm H₂O positive end-expiratory pressure (PEEP). One of the carotid arteries was cannulated and connected to a physiological pressure transducer (Memscap AS, Cleveland, NOR) to record the systemic arterial pressure and heart rate on a physiological recorder (Power Lab, AD Instruments Pty Ltd, AU). The left internal jugular vein was catheterized for administration of intravenous fluid and/or drugs. Lines were fixed in place and heparinized saline (0.15 M, 2 U/ml) was infused (1 ml/h) to maintain the patency of the arterial and vein line. Continuous electrocardiographic monitoring was performed. Body temperature was continuously monitored via a rectal probe and maintained at 37 °C with a thermostatically controlled plate. Baseline data for cardiac function were collected using small animal ultrasound, which is an animal-dedicated machine (VisualSonics Vevo® 2100 System; VisualSonics Inc, Toronto, Ontario, Canada).

Arterial blood pressure and heart rate were recorded with reference to atmospheric pressure at the mid-thoracic level at end expiration. All variables were displayed and collected on a personal computer.

Animals were randomly separated into either a control group (n = 15) or an ALI group (n = 15). ALI was induced by intravenous injection of oleic acid (OA) (Sigma-Aldrich, United Kingdom), which was prepared as a 1:1 mixture with pure ethanol [9]. The rats were placed in the supine position and infused via the left internal jugular vein with OA (9 ul/100 g) over 5 min. The control group was infused with an equal volume of 0.9 % normal saline (NS). Additional pentobarbital sodium (25 mg/kg) was administered every 45 min, providing a constant depth of anesthesia as monitored by the animal’s cardiorespiratory and reflex responses. Cardiac function was evaluated by echocardiography, and B lines were assessed by LUS before the experiment and at 1 h following the injection of OA/NS. Repeatability studies of the LUS technique were also conducted between the intra-observer and the inter-observer. The animals were then euthanized by exsanguination under anesthesia. Lungs were removed immediately post-mortem for further analysis. To confirm the presence of injury, lung tissue was collected for histologic analysis for three rats from each group.

LUS images were obtained using the small animal ultrasound, an animal-dedicated machine (VisualSonics Vevo® 2100 System; VisualSonics Inc, Toronto, Ontario, Canada) with the cardiac probe (30 MHz). In contrast to some other models, the rats were evaluated while in the prone position. Images were obtained from the dorsal wall in prone position (Fig. 1a). The reason for such a position was that heart is close to the anterior wall, and the beating heart may interfere with the quality of LUS images.

In humans, the 28-rib space technique is used to semi-quantitatively evaluate the volume EVLW [10], and the number of B-lines are counted at four sites (parasternal, mid-clavicular, anterior-axillary and mid-axillary lines) in each space from the second to the fourth intercostal spaces of the left hemithorax and the second to the fifth intercostal spaces on the right (3*4 + 4*4 = 28). This evaluation is performed at precisely defined lines for accurate measurements. In rats, the “28-rib space” technique was modified due to the smaller area of the rats’ backside. The LUS consisted of bilateral scanning of the posterior(dorsal)and lateral chest walls. Each hemithorax was divided into two zones: (1) from the posterior axillary line to the scapular line and (2) from the scapular line to the paravertebral line. A total of four scans were performed for each rat. Lung scanning was performed in the vertical plane on the rat’s dorsal wall (Fig. 1b). In each scan, five to six intercostal spaces were present on the screen. The probe was oriented in the “up and down” position to allow for whole lung imaging, which consistently demonstrated seven intercostal spaces (Fig. 1c). In summary, the lung was conceptually divided into four parts, and each parts contained seven intercostal spaces. This manner was analogous to the “28-rib space” evaluated in humans, with the only difference being that the vertical scanning plane was used in rats while the horizontal scanning plane is typically used in humans. The scanning sites were marked by a drawing pen in order to put the probe at exactly the same anatomical point each time.

For each scan, the A lines or B lines was recorded at baseline [11], and 1 h after the injection of OA or NS. The A lines were present as repetitive horizontal artifacts parallel to the pleural line and arising from it, caused by the preponderance of air over liquid in the lung parenchyma. The presence of A lines was given a score of zero, indicating a normal LUS pattern. The B lines defined as discrete laser-like vertical hyperechoic reverberation artifacts that arise from the pleural line (previously described as “comet tails”), extending to the bottom of the screen without fading, and move synchronously with lung sliding [10]. The B lines score was evaluated as follows: at each intercostal space, the number of B lines was counted (ranging from zero to ten, corresponding to a confluency of signal) and then the percentage of the rib space occupied by B-lines was determined and divided by ten (see Fig. 2). The B lines scores was evaluated by two physicians trained in the evaluation of chest ultrasound, and a consensus was reached when there was disagreement.

Blood samples were collected from abdominal aorta immediately after sacrifice and subjected to centrifugation (3000xG for 10 min). The resulting serum was frozen at−20 °C until biochemical analysis was performed. Biochemical markers (including hemoglobin concentration, haematocrit, and the wet/dry weight ratio of the blood) were measured in the serum by an automated biochemical analyzer (sysmex xs-800i, STAC Medical Science & Technology Co, Ltd,Japan). The lungs were removed immediately post-mortem, drained of blood and weighed. An equal amount of water was added to induce hemolysis. The lungs and the added water were homogenized using a commercial blender and an electric homogenizer. The dry weights of the blood, homogenate and supernatant were determined after drying at 60 °C for 72 h. After that, the fractional water contents were calculated and used to calculate the total lung water and the intravascular lung water. EVLW was defined as the difference between the total lung water and the intravascular lung water [12].

In each group, lung tissue of three rats as immersed in 10 % formaldehyde fixative for 24 h and then washed with water. For light microscopic examination, lung tissue was dehydrated with graded alcohol and then embedded in paraffin at 60 °C. Histologic sections were then stained with hematoxylin and eosin.

The inter-observer and intra-observer reproducibility of B lines score was assessed in all the ALI group. Both physicians are expert echocardiography technicians, one of them had received specific training on chest LUS, and the second one was trained by the first one (in a 2 h practical session) to measure the B lines score. The reliability of B lines score over time was investigated in a a series of 15 rats after LPS injection.

Hemodynamic and respiratory variables are shown in Table 1 for the two groups of animals, treated with NS or OA and euthanized after 1 h (Table 1).

Each assessment of B-lines was performed in less than 5 min, with full feasibility of 100 %. At baseline, the mean number of B-lines in the control group was 1.6 ± 0.8, after OA injection, and the number of B-lines increased bilaterally over time and more markedly at 1 h later with a mean of 15.8 ± 4.9 (Fig. 3).

The extravascular fluid content of the lung samples expressed by the wet-dry ratio significantly increased after the OA injection compared with the sham-operated control group (X vs. Y, p < 0.05). The correlation between the two methods of assessing EVLW is shown in Fig. 4 and provides an r = 0.834 (p < 0.001).

The histopathological changes between the NS (control) group and experimental group are shown in Fig. 5. Compared with the NS group, the experimental group demonstrated severe pulmonary hemorrhagic edema with inflammatory cell infiltration (Fig. 5).

The reproducibility of the B lines score over time in 15 rats was good in that the difference between the 2 measurements was always less than 2 SD of the average measurement (concordance index = 0.86, 95 % confidence interval: 0.62 to 0.96) (Fig. 6). The agreement between the expert and the naive observer was quite satisfactory: in over 15 independent measurements, only in 1 rats did the between-observer difference exceed 2SD (Fig. 6).