Study design
Regional ventilation distribution was measured in seven rats in each of four body positions (prone, supine, right- and left-lateral) in random order with all He3 MRI images obtained first, followed by EIT.
Animal preparation
The study was approved by the institutional ethics committee. Seven Wistar rats (8 to 10 weeks of age, 286 ± 21 g of either sex) were studied. The rats were anaesthetized, incubated and prepared as previously described
[8]. The chest was circumferentially shaved and 16 epicardial pacing wires (Medtronic Inc, Minneapolis, MN, USA) were sutured through the skin and the panniculosus carnosus with equal inter-electrode spacing
[8].
The rats were ventilated using a MRI compatible time-cycled, pressure-limited ventilator based on that of Hedlund
[9] with a respiratory rate of 80 breaths per minute and a tidal volume of ~10 mL/kg.
Hyperpolarized helium MRI (He3 MRI)
Rats were imaged with a Bruker (Ettlingen, Germany) 4.6 T ADVANCE spectrometer running Paravision 5.0 software. A 72 mm ID volume coil containing a 1 H (190.2 MHz) and 3He (144.9 MHz) RF coils was used. A single 40 mm 3He axial projection was acquired through the lungs with the following parameters: TR = 6 ms, TE = 1.7 ms, excitation pulse angle = 22°, field of view = 40 × 40 mm, acquisition matrix 96 × 60, centric phase encoding, scan time = 360 ms. The data was zero filled to give an image matrix of 128 × 88.
Three 2 ml breaths of He3 MRI were used to wash air from the lungs and increase the signal-to-noise ratio of the final image. Following washout, a 2 ml breath of HP3He was given with an inspiratory pause of 2 seconds. The images were acquired during this pause. The rat’s position was then changed and ventilation continued with air for at least 1 minute before the next He3 MRI image. The pacing wires used for EIT were removed prior to MR scanning but the scan position was adjusted to the same level on the chest as the EIT measurements were taken from. Switching of the inhaled tracer gas was achieved with computer controlled mechanical valves and gated to the MRI and it was not necessary to disconnect the animal from the ventilator.
Electrical impedance tomography (EIT)
A Göttingen GoeMF II EIT tomography (Sensor medics/VIASYS Healthcare, Netherlands) was used
[10]. For the EIT measurements, the rats were ventilated with 70% helium with 30% oxygen (Heliox). Measurements were taken at 44 frames per second with a 100 kHz, 5mApk-pk current. Data were processed as previously described
[6].
Data analysis
Image processing
The 128 × 88 pixel He3 MRI images were scaled
[11] to 1408 × 1408 pixels (lowest common multiple) in order to make individual pixels square, consistent with the EIT data. This scaling did not alter the aspect ratio of the image. The 32 × 32 pixel EIT images were scaled
[11] to 1408 × 1408 pixels without interpolation to produce an image size consistent with that of the 3He MRI. The EIT back projection algorithm assumes a round cross-section of the chest, which is not the case in rodents. The chest circumference of rodents has a relatively prominent sternum but the lungs within have a more or less round shape. To investigate the impact of this assumption of the EIT back projection algorithm and to compare the anatomical images given by HP3He MR imaging and EIT, the anterior to posterior and the left to right diameters (in pixels) were reported. The number of non-zero pixels was determined for each image.
As both techniques produce pixel-based images, two measures of regional ventilation distribution, commonly used in the analysis of EIT images, were employed to analyze both the He3 MRI and EIT data.
The geometric centre (GC) of each image was calculated using the “weighted centre of mass” function available in Imaged (free image analysis software provided by NIH)
[10]. The GC is a measure for ventilation distribution and identifies preferentially ventilated lung regions. This method is computationally equivalent to previously used methods
[12]. To allow comparison of different sized lung images the location of the GC was expressed as the percentage of the anterior to posterior and left to right aspect of the lung.
Tidal volume distribution, as a measure of ventilation in homogeneity, was quantified using the global in homogeneity index (GI)
[13]. The median value of all non-zero pixels in the image was calculated. The absolute difference between the median and each non-zero pixel was summed, and the sum normalized to the number of non-zero pixels. The lower the GI value to more homogeneous the ventilation is distributed.
Statistics
Two-way ANOVAs and t-tests were used as appropriate to compare parameters between positions and techniques. Data were described using mean and standard deviation. A P-value of <0.05 was considered significant. For statistical analyses GraphPad Prism pt?>3.02 (GraphPad Software, La Jolla, USA) was used. Significance was accepted at p < 0.05.