Name: 3D Cine Magnetic Resonance Imaging of Respiratory Motion in Mechanically Ventilated Mice and Rats (Video) | JoVE Journal
Uploaded: 2025-09-19T12:30:00.000+00:00
Duration: 8 min 22 s
Description: Learn 3D cine MRI of respiratory motion in mechanically ventilated mice and rats using accelerated motion-resolved imaging. This method reveals how PEEP affects diaphragm position and thoracic dynamics.

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5 minutes

This work was supported by ZonMW Grant 09120011910004&#160;(GS, CO, LH) and NIH grant 5R01HL121500 (CO).

<ol><li>Bassi, T., Taran, S., Girard, T. D., Robba, C., Goligher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ventilator-associated+brain+injury%3A+A+new+priority+for+research+in+mechanical+ventilation%5BTitle%5D)+AND+%22Am+J+Respir+Crit+Care+Med%22%5BJournal%5D)">Ventilator-associated brain injury: A new priority for research in mechanical ventilation.</a> Am J Respir Crit Care Med. 209 (10), 1186-1188 (2024).</li>
<li>Slutsky, A. S., Ranieri, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ventilator-induced+lung+injury%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">Ventilator-induced lung injury.</a> N Engl J Med. 369 (22), 2126-2136 (2013).</li>
<li>Levine, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+disuse+atrophy+of+diaphragm+fibers+in+mechanically+ventilated+humans%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">Rapid disuse atrophy of diaphragm fibers in mechanically ventilated humans.</a> N Engl J Med. 358 (13), 1327-1335 (2008).</li>
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<li>Damiani, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+reverse+triggering+dyssynchrony+during+lung-protective+ventilation+on+diaphragm+function%3A+An+experimental+model%5BTitle%5D)+AND+%22Am+J+Respir+Crit+Care+Med%22%5BJournal%5D)">Impact of reverse triggering dyssynchrony during lung-protective ventilation on diaphragm function: An experimental model.</a> Am J Respir Crit Care Med. 205 (6), 663-673 (2022).</li>
<li>Guillon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preclinical+septic+shock+research%3A+why+we+need+an+animal+ICU%5BTitle%5D)+AND+%22Ann+Intensive+Care%22%5BJournal%5D)">Preclinical septic shock research: why we need an animal ICU.</a> Ann Intensive Care. 9 (1), 66(2019).</li>
<li>Larsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+animal+models+of+muscle+wasting+in+intensive+care+unit+patients%5BTitle%5D)+AND+%22Crit+Care+Med%22%5BJournal%5D)">Experimental animal models of muscle wasting in intensive care unit patients.</a> Crit Care Med. 35 (9), S484-S487 (2007).</li>
<li>Schwarte, L. A., Zuurbier, C. J., Ince, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+ventilation+of+mice%5BTitle%5D)+AND+%22Basic+Res+Cardiol%22%5BJournal%5D)">Mechanical ventilation of mice.</a> Basic Res Cardiol. 95 (6), 510-520 (2000).</li>
<li>Leith, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+mammalian+respiratory+mechanics%5BTitle%5D)+AND+%22Am+Rev+Respir+Dis%22%5BJournal%5D)">Comparative mammalian respiratory mechanics.</a> Am Rev Respir Dis. 128 (2 Pt 2), S77-S82 (1983).</li>
<li>Vinegar, A., Sinnett, E. E., Leith, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+mechanisms+determine+functional+residual+capacity+in+mice%2C+Mus+musculus%5BTitle%5D)+AND+%22J+Appl+Physiol+Respir+Environ+Exerc+Physiol%22%5BJournal%5D)">Dynamic mechanisms determine functional residual capacity in mice, Mus musculus.</a> J Appl Physiol Respir Environ Exerc Physiol. 46 (5), 867-871 (1979).</li>
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<li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Technical+aspects+of+small+animal+CMR+imaging%5BTitle%5D)+AND+%22Front+Phys%22%5BJournal%5D)">Technical aspects of small animal CMR imaging.</a> Front Phys. 8, ARTN 183(2020).</li>
<li>Daal, M. R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+mouse+heart+left+ventricular+function%2C+myocardial+strain%2C+and+hemodynamic+forces+by+cardiovascular+magnetic+resonance+imaging%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Quantification of mouse heart left ventricular function, myocardial strain, and hemodynamic forces by cardiovascular magnetic resonance imaging.</a> J Vis Exp. (171), e62595(2021).</li>
<li>Gottwald, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudo-spiral+sampling+and+compressed+sensing+reconstruction+provides+flexibility+of+temporal+resolution+in+accelerated+aortic+4D+flow+MRI%3A+A+comparison+with+k-t+principal+component+analysis%5BTitle%5D)+AND+%22NMR+Biomed%22%5BJournal%5D)">Pseudo-spiral sampling and compressed sensing reconstruction provides flexibility of temporal resolution in accelerated aortic 4D flow MRI: A comparison with k-t principal component analysis.</a> NMR Biomed. 33 (4), e4255(2020).</li>
<li>Sudy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Different+contributions+from+lungs+and+chest+wall+to+respiratory+mechanics+in+mice%2C+rats%2C+and+rabbits%5BTitle%5D)+AND+%22J+Appl+Physiol+%281985%29%22%5BJournal%5D)">Different contributions from lungs and chest wall to respiratory mechanics in mice, rats, and rabbits.</a> J Appl Physiol (1985). 127 (1), 198-204 (2019).</li>
<li>Strijkers, G. J. Retrospective-v9.7: Self-gated cine MRI reconstruction. , Available from: 
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<li>Caro, A. C., Hankenson, F. C., Marx, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+thermoregulatory+devices+used+during+anesthesia+of+C57BL%2F6+mice+and+correlations+between+body+temperature+and+physiologic+parameters%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Comparison of thermoregulatory devices used during anesthesia of C57BL/6 mice and correlations between body temperature and physiologic parameters.</a> J Am Assoc Lab Anim Sci. 52 (5), 577-583 (2013).</li>
<li>Tang, M., Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progress+in+Understanding+Radiofrequency+Heating+and+Burn+Injuries+for+Safer+MR+Imaging%5BTitle%5D)+AND+%22Magn+Reson+Med+Sci%22%5BJournal%5D)">Progress in Understanding Radiofrequency Heating and Burn Injuries for Safer MR Imaging.</a> Magn Reson Med Sci. 22 (1), 7-25 (2023).</li>
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Ruslan Garipov is an employee of MR Solutions Ltd., Guildford, United Kingdom.

This study shows that 3D MR imaging with retrospective gating allows to measure thorax movement in two animal models during mechanical ventilation. These techniques have the potential to be used in future research involving cardiac and thoracic imaging in mechanically ventilated mice and rats. We found that due to the fixed respiration frequency, retrospective binning was highly robust.
A critical step of the protocol is setting up the correct positioning of the animals within the MR coils. During scanning, it is not possible to reposition the animals. Additionally, maintaining normothermia is very important to ensure that measurements are made under physiological conditions, as anesthetics can cause hypothermia through peripheral vasodilation, and the radiofrequency pulses of this MRI protocol can cause hyperthermia17,18. In Figure 7, body temperature fluctuations can be seen during the short period around the transfer of the animals to the scanner (around the 1 h mark) in which no heat is applied from an external source. Another critical step is the titration of anesthesia, especially in mice. The plane of anesthesia where the mice do not exhibit respiratory effort but maintain stable vital parameters is narrow and requires careful monitoring of the respiratory signal and adjustment of anesthesia infusion rates. In rats, we did not face such issues within the range of sedation used. The image quality will be negatively impacted if there are respiratory efforts of the animal because of the retrospective reconstruction of the images based on the set respiratory frequency. It is possible to use a different anesthetic regimen, such as inhalation anesthesia with isoflurane or alternative injection anesthetics that cause more suppression of respiratory effort. The CWE-MRI-1 small animal ventilator used in this protocol is compatible with inhalation anesthetics19.
There are three critical steps for reproducing the MRI method proposed in this study. First, the same basic MRI sequence (i.e., 3D FLASH) must be used. Such a sequence is available on basically all MRI systems. Secondly, the k-space trajectory mentioned in this work (pseudo-spiral) may require a software patch that can execute user-defined k-space trajectories. We invite readers to reach out to the corresponding author if they wish to replicate the acquisition. There is experience in implementing this type of acquisition on systems from other vendors. Lastly, the reconstruction pipeline that we share as open-source software is vendor-neutral and, therefore, applicable to all preclinical systems.
We chose retrospective triggering and binning over prospective triggering for several reasons. Primarily, retrospective triggering and k-space data binning offer full flexibility in the number of reconstructed respiratory CINE frames, allowing us to optimize temporal resolution, spatial resolution, and signal-to-noise ratio. Additionally, prospective triggering introduces a dead time at the end of each respiratory cycle as the scanner waits for the next trigger, which can disrupt the steady state and result in an incomplete cycle. With mechanical ventilation, this dead time is minimal but still present.
The main limitation of this protocol is the inability to measure airway or esophageal pressures. To our knowledge, there are no MR-compatible lung&#160;or esophageal pressure sensors for small animals on the market. Another limitation is the lack of continuous blood pressure measurements within the MRI. The anesthetic protocol was based on prior institutional research. Atropine was added because of the high prevalence of bradycardia and hypotension observed previously. Although continuous blood pressure monitoring was not possible due to MR-environment constraints, hypertension is unlikely, as no tachycardia was observed (Figure 7). The monitoring employed in this protocol consisted of heart rate, oxygen saturation, temperature, and a respiratory signal based on movement of the thoracic wall. Using these measurements, an overview of the vital parameters of the animals can guide the administration of anesthesia but having a blood pressure read could prevent overdosing animals showing respiratory effort. No other MRI studies concerning the respiratory physiology of mechanically ventilated rodents have been published, but this method can be employed for a wide range of research questions outside of respiratory research. The reconstruction program mentioned in the manuscript (Retrospective) can also be used to reconstruct images throughout the cardiac cycle, thus allowing the researcher to acquire high-resolution images of the heart and vessels without motion artifacts. This protocol can be applied in every project that requires mechanical ventilation of rats or mice while scanning. One can, for example, consider studying the relation between mechanical ventilation settings and cardiac function. Other options include imaging animals with an open chest or after surgeries requiring mechanical ventilation.

Mechanical ventilation is the cornerstone of treatment for acute respiratory failure in the intensive care unit. Unfortunately, it can also cause injury. Ventilator-induced lung injury, ventilator-associated brain injury, and ventilator-induced diaphragm dysfunction are examples of iatrogenic complications associated with acute mechanical ventilation1,2,3. The pathophysiology underlying these mechanisms of injury remains poorly understood. Experimental work in animals has identified important mechanisms that underlie complications of mechanical ventilation, allowing for translation to clinical practice through the application of new ventilation strategies4. A recent example is the research on the impact of ventilator dyssynchronies on diaphragm function in porcine ARDS models5. Such research in mechanically ventilated animal models is vital to elucidate the mechanisms of injury, even with the current tendency to move away from animal testing. Studies on the effects of mechanical ventilation require an intact cardiopulmonary system and the ability to simulate pathophysiology commonly seen in the ICU4. Large animal models are used often because they mimic human physiology more closely than rodents and can be ventilated with machines that are used in the ICU4. The main drawbacks are the large cost associated with using large animals and the lack of genetically engineered large animal models to investigate molecular mechanisms of disease6,7. Therefore, experiments in mechanically ventilated rodents are unavoidable as long as alternatives, such as sophisticated tissue cultures or organ-on-a-chip models, are not available.
Various interventions that improved outcomes in animals did not translate to humans4,6, underlining the importance of understanding how commonly used animal models of mechanical ventilation differ from humans. The effects of ventilator settings on the respiratory system are determined by a plethora of factors that may be species-specific, such as tissue elastance, muscle tone, and variation in the response to commonly used anesthetics8,9,10,11. These differences may lead to contrasting effects on thorax and lung movement and geometry that may account for the disparity between humans and animals. Investigating the effect of ventilation settings on the geometry and movement of the cardiopulmonary system may elucidate differences between species and may aid in the translation of pathophysiological concepts derived from animal studies to the clinical setting. The technical advancement of motion-resolved cardiovascular magnetic resonance imaging has enabled imaging of the beating heart in rodent models, providing insights into shape, function, and pathophysiology in rodent models of cardiovascular disease12,13. These methods are underutilized to image the thorax and movement during the respiratory cycle in mechanically ventilated animals.
Here, we present a method to study thorax geometry and movements in mechanically ventilated rodents using 3D cine MRI. This allows for studying the acute effects of numerous ventilator settings or other interventions on thorax geometry and movement in mechanically ventilated rodents. We applied a novel pseudo-spiral, respiratory-gated 3D cine MRI acquisition for ventilated small animals13,14. In this initial study, we investigated the effects of positive-end-expiratory pressure (PEEP), but this method can also be used to study the effect of other interventions or respiratory settings, such as I: E ratio, tidal volume, and respiratory rate. This method will open up a range of possibilities for pre-clinical imaging of rodent models that require mechanical ventilation in the field of intensive care research and beyond.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;11 Plus Digital Dual Syringe Pump</td><td>Harvard apparatus (USA)</td><td>229193-2</td><td>dual syringe pump with programmable features for laboratories</td></tr><tr><td>AccuSens single and multi-channel signal conditioner</td><td>Opsens solutions inc. (Canada)</td><td>ACS-P4-N-62SC</td><td>Used with fiber optic temperature sensor to monitor body temperature</td></tr><tr><td>Fiber optic temperature sensor&amp;nbsp;</td><td>Opsens solutions inc. (Canada)</td><td>OTP-M</td><td></td></tr><tr><td>Mouse cradle</td><td>&amp;Eacute;quipment V&amp;eacute;t&amp;eacute;rinaire Minerve (France)</td><td></td><td></td></tr><tr><td>Mousestat Jr.&amp;nbsp;</td><td>Kent-scientific (USA)</td><td>MSTAT-JR</td><td>The MouseSTAT Jr. provides&amp;nbsp; accurate pulse oximetry in both mice and rats.</td></tr><tr><td>MR Scanner</td><td>MR Solutions Lt. (United Kingdom)</td><td>MRS-7024</td><td></td></tr><tr><td>MR-compatible Monitoring &amp;amp; Gating System for Small animals</td><td>SA Instruments, Inc. (USA)</td><td>1030</td><td>RT Module (respiratory interface module) , ERT Control/ Gating Module, battery pack</td></tr><tr><td>MRI-1 compatible ventilator</td><td>CWE (USA)</td><td>12-07000</td><td>Mechanical ventilator suitable for ventilating small animals in magnetic resonance environments. It ventilates with a volume-controlled, flow x time based principle using pneumatic valves.</td></tr><tr><td>Multistation temperature control unit and High Flow PCA</td><td>&amp;Eacute;quipment V&amp;eacute;t&amp;eacute;rinaire Minerve (France)</td><td></td><td></td></tr><tr><td>PEEP column</td><td></td><td></td><td>Repurposed fluid measuring column</td></tr><tr><td>Physiosuite Mechanical Ventilator</td><td>Kent-scientific (USA)</td><td></td><td>Mechanical ventilator suitable for ventilating small animals. It ventilates with a volume-controlled, pressure-regulated principle.&amp;nbsp;</td></tr><tr><td>Pulse Oximeter Paw Sensors, MRI-Compatible</td><td>Kent-scientific (USA)</td><td>SPO2-MSE-MRI</td><td>MRI-compatible paw sensor for Mousestat Jr.</td></tr><tr><td>Rat cradle</td><td>&amp;Eacute;quipment V&amp;eacute;t&amp;eacute;rinaire Minerve (France)</td><td></td><td></td></tr><tr><td>RF coil, Mouse</td><td>MR Solutions Lt. (United Kingdom)</td><td></td><td>38mm mouse volume RF coil for mouse body studies</td></tr><tr><td>RF coil, Rat</td><td>MR Solutions Lt. (United Kingdom)</td><td></td><td>65mm&amp;nbsp; rat volume RF coil for rat body studies</td></tr><tr><td>&lt;strong&gt;Surgical tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Abbocath 14G, 20G, 22G, 24G</td><td>Abbocath</td><td></td><td>14G, 20G, 22G for use as endotracheal tube, 24G for intraperitoneal infusion of anesthesia</td></tr><tr><td>Ethilon 3-0&amp;nbsp; FD-1 suture with needle</td><td>Ethicon (USA)</td><td>663H</td><td></td></tr><tr><td>Forceps #55</td><td>Dumont (Switzerland)</td><td>55</td><td></td></tr><tr><td>Metzenbaum Scissors</td><td>Fine science tools (Germany)</td><td>14017-14</td><td></td></tr><tr><td>Needle holder</td><td>Fine science tools (Germany)</td><td>12005-15</td><td></td></tr><tr><td>Nonabsorbable braided silk suture 6/0</td><td>Fine science tools (Germany)</td><td>18020-60</td><td></td></tr><tr><td>Spring Scissors</td><td>Fine science tools (Germany)</td><td>15033-09</td><td></td></tr><tr><td>Stevens tenotomy scissors</td><td>Fine science tools (Germany)</td><td>14064-11</td><td></td></tr><tr><td>Suture Tying Forceps</td><td>Fine science tools (Germany)</td><td>18025-10</td><td></td></tr><tr><td>Tape medipore</td><td>3M (USA)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Medication&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Atropine 0.25 mg/mL</td><td></td><td></td><td></td></tr><tr><td>Dexmedetomidine 0.5 mg/mL Dexdormitor</td><td>Orion pharma (Finland)</td><td></td><td></td></tr><tr><td>Duratears eye ointment</td><td>Alcon Nederland BV (Netherlands)</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>AST pharma (France)</td><td></td><td></td></tr><tr><td>Ketamine 100 mg/mL Ketexx</td><td>Alfasan (Netherlands)</td><td></td><td></td></tr><tr><td>Vaseline petroleum jelly</td><td>Unilever (Netherlands)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Elastix</td><td>Image Sciences Institute, UMC Utrecht (Netherlands)</td><td></td><td></td></tr><tr><td>Itksnap</td><td>University of Pennsylvania (USA)</td><td></td><td></td></tr><tr><td>Matlab 2019a</td><td>Wolfram Research Inc (USA)</td><td></td><td></td></tr><tr><td>Microdicom</td><td>Microdicom (Bulgaria)</td><td></td><td></td></tr><tr><td>Retropective</td><td>Amsterdam UMC (Netherlands)</td><td></td><td></td></tr></tbody></table>

3d cine magnetic resonance imaging of respiratory motion in mechanically ventilated mice and rats

3D Cine Magnetic Resonance Imaging of Respiratory Motion in Mechanically Ventilated Mice and Rats

The pathophysiology of diaphragm dysfunction in mechanically ventilated patients has yet to be fully understood, and adequate animal models are required to accommodate further research. Mechanical ventilation settings, such as the level of positive end-expiratory pressure (PEEP), play a crucial role. The goal was to develop a method to image 3D thoracic movement during mechanical ventilation of mice and rats at different respiratory pressure settings. Rats (Wistar) and mice (C57BL/6) were anesthetized with a mix of ketamine, atropine, and dexmedetomidine. Anesthesia was maintained by continuous infusion through a peritoneal catheter. Next, a tracheostomy was performed to enable mechanical ventilation of the animals. Animals were placed in a 7T MR system while ventilated with an MR-compatible ventilator. 3D cine imaging of the thorax was conducted using a 3D gradient echo sequence with pseudo-spiral k-space filling. A navigator signal, generated by the slice selection rewinder gradient, was recorded every TR. Retrospective binning and reconstruction of the data in 12 respiratory cine time frames was performed using in-house developed software. We successfully visualized thoracic movement in 3D in both species during mechanical ventilation, enabling the investigation of changes in thorax geometry throughout the respiratory cycle at varying PEEP levels. We found that retrospective binning of respiratory frames was highly facilitated by the fixed respiration rate. The protocol presented here can be used to study cardiac and thoracic geometry and movement in mechanically ventilated mice and rats.

Using the protocol described in this study, we scanned a group of 8 healthy C57BL/6 WT mice and 8 Wistar WT rats successfully using a 7 Tesla MRI scanner. We were able to mechanically ventilate the animals at different levels of PEEP and monitor basic physiological parameters during scanning (Figure 7).
In Figure 5, an example of three 2D scans in the end-expiratory frame is shown of a mouse and a rat in sagittal orientation. As can be seen, PEEP introduces a caudal displacement of the diaphragm in both animals, but in mice, this is accompanied by a widening of the thorax as well, indicated by an increase in the anterior-posterior diameter.
These high-quality scans show that using this technique, we can measure the size of the different parts of the thorax and show the effects of various ventilatory settings on the movement of the thorax and the diaphragm. The short scan time allows for within-subjects comparison of the outcome parameters that one is interested in, reducing the required number of animals. Table 2 shows the outcome parameters of all animals.
Figure 8 shows the lung segmentations of the end-expiratory dynamic at three PEEP levels with the corresponding lung volume. As expected, increasing PEEP resulted in an increase in lung volume. The 3D cines are also displayed as a movie in Supplementary Video 1 and Supplementary Video 2.
<img alt="MRI analysis software interface; graph plots signal data, heart rate, respiration, reconstructed images." class="xfigimg" src="/files/ftp_upload/67474/67474fig04.jpg" data-altupdatedat="1762425657000" data-alt="Figure 4"> 
Figure 4: Screenshot of the software used to reconstruct the images with retrospective gating of the data. The gating of the data is based on the navigator signal. <a href="https://www.jove.com/files/ftp_upload/67474/67474fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="MRI lung measurements in rats and mice under different PEEP conditions; anatomical comparison chart." class="xfigimg" src="/files/ftp_upload/67474/67474fig05.jpg" data-altupdatedat="1762425657000" data-alt="Figure 5"> 
Figure 5: Representative results of 2D images in a rat and a mouse at three PEEP levels, with the anterior-posterior and cranio-caudal lung dimensions. <a href="https://www.jove.com/files/ftp_upload/67474/67474fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="MRI segmentation analysis diagram; displays red-highlighted organ structure, axial and sagittal views." class="xfigimg" src="/files/ftp_upload/67474/67474fig06.jpg" data-altupdatedat="1762425657000" data-alt="Figure 6"> 
Figure 6: Screenshot of the software used to segment the lungs in the 3D cines. In the left-top panel, an axial view of the 3D cine is shown with the lung segmented in red. In the right-top panel a sagittal view is shown with the lung segmented in red. In the left bottom panel a 3D render of the segmented lung is shown. In the left-right panel, a coronal view is shown with the lung segmented in red. <a href="https://www.jove.com/files/ftp_upload/67474/67474fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cardiorespiratory data comparison: Heart rate, temperature, O2 saturation, mouse vs. rat, time-series graph." class="xfigimg" src="/files/ftp_upload/67474/67474fig07.jpg" data-altupdatedat="1762425657000" data-alt="Figure 7"> 
Figure 7: Vital parameters of the animals during the experiments. N = 8 mice (blue), N = 8 Rats (orange). Data is shown as mean with SD. Note the drop in heart rate when the animals enter a surgical plane of anesthesia. <a href="https://www.jove.com/files/ftp_upload/67474/67474fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="PEEP effect on lung volume, 3D model, anterior/posterior, respiratory mechanics, CT scan analysis." class="xfigimg" src="/files/ftp_upload/67474/67474fig08.jpg" data-altupdatedat="1762425657000" data-alt="Figure 8"> 
Figure 8: 3D Segmentation of the lungs at three PEEP levels with the corresponding lung volumes. Segmentation of a rat lung at PEEP 0 (left column), PEEP 3 (middle column), and PEEP 6 (right column). In the top row, the posterior side of the lungs is shown. In the middle row the anterior side of the lungs is shown. The volumes that are shown in the bottom row are measured within the 3D-rendered lungs that are visible in this figure. <a href="https://www.jove.com/files/ftp_upload/67474/67474fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video 1: 3D cine of a mouse. <a href="https://www.jove.com/files/ftp_upload/67474/Supplemental_Video1.gif" target="_blank">Please click here to download this Video.</a>
Supplementary Video 2: 3D cine of a rat. <a href="https://www.jove.com/files/ftp_upload/67474/Supplemental_Video2.gif" target="_blank">Please click here to download this Video.</a>
Supplementary Coding File 1: Script to register the segmentations to other acquisitions. <a href="https://www.jove.com/files/ftp_upload/67474/Supplemental_Video1.gif" target="_blank">Please click here to download this File.</a>

The proposed setup allows controlled mechanical ventilation and Magnetic Resonance imaging of 3D thorax movement in mice and rats. We have used this setup to study the pathophysiology underlying mechanical ventilation-induced respiratory muscle dysfunction.

The pathophysiology of diaphragm dysfunction in mechanically ventilated patients has yet to be fully understood, and adequate animal models are required ...

PEEP is the positive end-expiratory pressure applied by a mechanical ventilator at the end of a breath. We wanted to explore how the ventilator settings, particularly PEEP, affect the shape and movement of the thorax. We compare the results between mice and rats.Currently, mainly ultrasound is used for visualizing ventilated animals. With accelerated motion-resolved cine imaging using 70 MRI in combination with an optimized anesthesia protocol, we successfully performed 3D imaging of mechanically ventilated mice and rats. So from the MR images, we segmented the lungs to study the movement and dimension of the thorax.And we can, for example, assess if the thorax expands more in the craniocaudal or in the anterior-posterior direction. Our main focus lies on preventing diaphragm and lung injury in mechanically ventilated patients. In this project, we specifically study the parameter PEEP, but in the future, we also want to study other settings of the mechanical ventilator or use different types of genetically modified animal models.To begin, calibrate the mechanical ventilator for use outside the MR room using the ventilator menu. Place the mechanical ventilator in the magnetic resonance or MR room. Pull the cables connected to the MR-compatible ventilator through the bore of the MR machine.Next, connect the MR-compatible mechanical ventilator to the compressor and a source of oxygen flow. To prepare a positive end expiratory pressure or PEEP column, fill a 50-milliliter measuring column with water. Now, tape a respiratory cushion loosely to the MR cradle at the location where the thorax of the animal will be.Position the anesthetized and tracheostomized animal supine in the MR cradle and place the appropriate magnetic resonance coil over it, ensuring the tracheotomy tube is properly positioned. Stop and disconnect the mechanical ventilator and anesthesia pump. Transport the animal to the MR room and reconnect it to the MR-compatible ventilator to start ventilating.Insert the rectal thermometer probe. Place the sensor of the saturation and heart rate monitor on the hind limb of the animal. Connect the respiratory monitoring system to the respiratory cushion to ensure proper vitals.Place the animal in the MR scanner with the coil centered over the thorax. And connect the radio frequency cables to the coil. Immerse the expiratory tubing six centimeters into the water column, administering six centimeters of water PEEP.Acquire a scout with five images in coronal and axial orientations, ensuring that the full thorax is within the field of view. Then acquire the 2D scans at six centimeters of water PEEP. Obtain 3D scan after placing the field of view around the complete thorax to cover the 2D scans.After completing all the required scans, remove the animal from the scanner. To begin, obtain 2D and 3D MRI scans of the animal under mechanical ventilation, while applying positive end-expiratory pressure or PEEP. Open the software and click Load MRD to load the raw files.Enter the respiratory rate set on the MR-compatible mechanical ventilator into heart respiration filters, and set the width to 15. Press Filter to apply the settings, resulting in a gated navigator signal. Ensure that the red signal in the respiration navigator and respiration rate boxes follow the blue raw navigator signal.Select Respiratory Movie in the Cine tab, set the number of frames to 12 and the number of dynamics to one. In the reco parameters tab, enable PCA level eight and ringing filter. After applying the settings, press Reconstruct to initiate the reconstruction.Check the reconstructed images and export them with the export DCM feature. For image analysis, open and select the desired frame of the 2D image in the DICOM viewer. Using the measurement button in microdicom, measure the anterior-posterior and craniocaudal lung dimensions in the sagittal 2D cine, and the left-right dimension in the coronal-oriented 2D cine.After repeating these measurements for all PEEP levels, load the cine into the 3D image analysis program, reorient image under the Tools tab, and fill in SPL as RAI code. Scroll through the dynamics at tools followed by layer inspector and select the end-expiratory frame. Then, using paintbrush mode, initialize segmentation by manually contouring the middle part of the lungs in two slices for every orientation.Start the 3D segmentation using the active contour function in the main toolbar. Enable initialize with current segmentation and press Segment 3D after ensuring complete thorax coverage. Select thresholding as the pres segmentation mode.Use only an upper threshold with the right-most threshold mode, and press Next. Start active contouring with the play button. Pause it as soon as the segmentation reaches the edges of the lungs.After verifying the images, press Finish to finalize. Now, press update to visualize the 3D segmentation and rotate the 3D visualization to inspect. To save the segmentation as a nifty file, click Segmentation followed by Save Segmentation Image.Export the segmentation as an STL mesh file with the export segmentation mesh option. After noting the lung volumes, register the images of other respiratory states to the end-expiratory dynamic using the appropriate script, and apply the computed transformations to the segmentation. The high-quality MRI scans allowed monitoring of basic physiological parameters during mechanical ventilation of the animals at different levels of PEEP.The sagittal orientation scans demonstrated that PEEP caused caudal displacement of the diaphragm in both mouse and rat models, with an accompanying thorax widening observed exclusively in mice. Increasing PEEP levels in mice and rats was associated with measurable increases in lung volume, with values rising from 12.7 milliliters at zero PEEP to around 15.8 milliliters at six-centimeter water PEEP.

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