3R-Project 82-02

Magnetic Resonance Imaging (MRI) for the non-invasive assessment of lung inflammation and pulmonary function in the rat

Nicolau Beckmann
Novartis Institutes for BioMedical Research (NIBR), Postfach, 4056 Basel, Switzerland.
nicolau.beckmann@novartis.com

Keywords: rat; lung; asthma; inflammation; magnetic resonance imaging (mri); non-invasive; reduction; refinement; drug screening

Duration: 2 years Project Completion: 2010

Background and Aim
Animal models and methods currently used to evaluate the efficacy of potential treatments for diseases of the airways are limited, generally invasive and terminal. The present project explores the flexibility of magnetic resonance imaging (MRI) to obtain anatomical and functional information of the lung, with the aim of developing a non-invasive approach to carry out drug screening in rat models of asthma and chronic obstructive pulmonary disease (COPD). Using MRI, the disease progression can be followed in a single animal. Thus, a significant reduction in the number of animals used for experimentation may be achieved, as well as minimal interference with their well-being and physiological status. Also, MRI has the potential to shorten the overall duration of the observation period after disease onset since the technique is able to detect changes induced by allergen before these are reflected in secreted parameters of inflammation.
Non-invasive MRI techniques shall be developed in order to
· assess pulmonary function (lung ventilation, perfusion)
· determine the pathophysiologic effects of chronic inflammation on the structure of lung tissues, e.g. airway remodeling and angiogenesis
· establish experimental protocols to measure the effects of drugs in both a) preventing and b) treating pulmonary inflammation in rat models of airway diseases

Method and Results
Procedures are described in detail in [1-3]. In brief:
1) Inflammation models: Two models of pulmonary inflammation will be studied by MRI: (i) Allergen-induced pulmonary inflammation in pre-sensitized rats, resulting from the intra-tracheal (i.t.) administration of ovalbumin (OVA). (ii) Endotoxin-induced pulmonary inflammation in non-sensitized rats, induced by instillation of lipopolysaccharide (LPS).
2) MRI: Measurements will be carried out with a spectrometer operating at 4.7 T. Animals will breath spontaneously during image acquisition.
3) Analysis of BAL fluid or Histology: These (terminal) methods will be used during the validation phase of the study to better characterize the signals detected by MRI and calibrate them against established methods of detecting lung damage. The lungs are lavaged and several inflammation parameters are assessed in the BAL, e.g. eosinophil and neutrophil numbers. Histology is carried out to assess e.g. perivascular edema and macrophage infiltration.

Conclusions and Relevance for 3R
The main advantage of using MRI as analysis tool in animal models of diseases is its non-invasive nature, which allows repeated measurements to be carried out in the same animal. This is of relevance in longitudinal studies since the inter-individual variance is eliminated and the number of animals to reach statistical significance is much smaller. In the case of rat models of asthma, we estimate we can reduce the number of animals used by approximately 80-90% as compared to conventional approaches [broncho-alveolar lavage (BAL) fluid analysis; bronchial biopsies; histology; weighing of lungs].

Since most of the studies are conducted on spontaneously breathing rats, the well-being of the animals during experimentation is improved, as invasive procedures like tracheotomy and/or intubation are avoided. Thus, repeated measurements can be carried out more easily, and the information obtained from time courses shall provide a better picture of disease development and treatment. A further aim is to replace current methods of ventilation assessment involving the use of radioactive materials. Finally, we estimate that, by using MRI, the duration of the experimental period can be reduced in some of the applications as compared to conventional approaches.

Non-invasiveness is a major asset when studying chronic diseases. The flexibility of MRI makes it a widely applicable method. We are currently using MRI techniques to assess drug effects non-invasively in several models of diseases, e.g. arthritis, transplantation, neuro- degeneration and stroke. For more details, please see [4].

Outlook:
The established protocols will be useful in characterising new compounds intended to prevent/resolving inflammation in asthma and chronic obstructive pulmonary disease (COPD). Of special interest is the non-invasive detection of pathophysiologic changes associated with chronic inflammation in these models. Furthermore, we intend to develop procedures that allow lung function to be measured non-invasively, thus allowing questions concerning the functionality of the lung after therapy to be addressed. Also, it is expected that by using MRI as analysis tool the overall duration of the observation period after disease onset can be shortened since the technique can detect changes induced by allergens before these are reflected in secreted parameters of inflammation.

References
1. Beckmann N, Tigani B, Ekatodramis D, Borer R, Mazzoni L, Fozard JR (2001). Pulmonary edema induced by allergen challenge in the rat: Noninvasive assessment by magnetic resonance imaging. Magn Reson Med 45, 88-95.

2. Beckmann N, Tigani B, Sugar R, Jackson AD, Jones G, Mazzoni L, Fozard JR (2002). Non-invasive detection of endotoxin induced mucus hypersecretion in rat lung by magnetic resonance imaging. Am J Physiol Lung Cell Mol Physiol 283, L22-L30.

3. Tigani B, Schaeublin E, Sugar R, Jackson AD, Fozard JR, Beckmann N (2002). Pulmonary inflammation monitored non-invasively by MRI in freely breathing rats. Biochem Biophys Res Commun 292, 216-221.

4. Beckmann N, Mueggler T, Allegrini PR, Laurent D, Rudin M (2001). From Anatomy to the Target: Contributions of Magnetic Resonance Imaging to Pre-Clinical Pharmaceutical Research . Anat Rec (New Anat) 265, 85-100.

5. Tigani B, Cannet C, Zurbrügg S, Schaeublin E, Mazzoni L, Fozard JR, Beckmann N. (2003) Resolution of the oedema associated with allergic pulmonary inflammation in rats assessed non-invasively by magnetic resonance imaging. Br J Pharmacol 140:239-246.

6. Beckmann N, Tigani B, Mazzoni L, Fozard R, (2003) Techniques: Magnetic resonance imaging of the lung provides potential for non-invasive preclinical evaluation of drugs. Trends Pharmacological Sciences 24/10, 550-554.

7. Beckmann N, Cannet C, Zurbruegg S, Rudin M, Tigani B (2004) Proton MRI of lung parenchyma reflects allergen-induced airway remodeling and endotoxin-aroused hyporesponsivness: A step toward ventilation studies in spontaneously breathing rats. Magn Reson Med 52, 258-268.

8. Beckmann N, Laurent D, Tigani B, Panizzutti R, Rudin M, (2004) Magnetic resonsnace imaging in drug discovery: lessons from disease areas. DDT 9, 35–42.

9. Tigani B, Gremlich UU, Cannet C, Zurbruegg S, Quintana HK, Beckmann N (2004). Near-infrared fluorescence imaging and histology confirm anomaöous edematous signal distribution detected in the rat lung by MRI after allergen challenge. J. Magn Res Imaging 20, 967-974.

10. Signor L, Tigani B, Beckmann N, Falchetto R, Stöckli M (2004) Two-dimensional electrophoresis protein profiling and identification in rat bronchoalveolar lavage fluid following allergen and endotoxin challenge. Proteomics 4, 2101-2110.

11. Beckmann N, Cremillieux Y, Tigani B, Quintanta HK, Ble FX, Fozard JR (2006) Lung MRI in small rodents as a tool for the evaluation of drugs in models of airways diseases. In: "In Vivo MR techniques in Drug Discovery and Development" (Editor: N.Beckmann), pp.351-372. Taylor & Francis, New York (2006).

12. Quintana HK., Cannet C., Schaeublin E., Zurbruegg S., Sugar R., Mazzoni L., Page C.P., Fozard J.R., and Beckmann N. (2006) Identification with MRI of the pleura as a major site of the acute inflammatory effects induced by ovalbumin and endotoxin challenge in the airways of the rat.Am J Physiol Lung Cell Mol Physiol 291: L651-L657.

13. Quintana HK, Cannet C, Zurbruegg S, Ble FX, Fozard JR, Page CP, Beckmann N. (2006) Proton MRI as a noninvasive tool to assess elastase-induced lung damage in spontaneously breathing rats.Magn Reson Med.56(6):1242-50.

14. Beckmann N, Quintanta HK, Ble FX, Cannet C.,Tigani B, Zurbruegg S, Fozard JR (2006) Magnetic Resonance Imaging (MRI) of the lung as a tool for the non-invasive evaluation of drugs in rat models of airways diseases. ALTEX 23, 420-428.

15. Karmouty Quintana, H., Cannet, C., Zurbruegg, S., Blé, F.X,, Fozard, J.R., Page, C.P., Beckmann, N. (2006). Proton MRI as a non-invasive tool to assess elastase-induced lung damage in spontaneously breathing rats. Magn Reson Med 56, 1242-1250.

16. Tigani, B., Cannet, C., Karmouty-Quintana, H., Ble, F.X., Zurbruegg, S., Schaeublin, E., Fozard, J.R., Beckmann, N. (2007). Lung inflammation and vascular remodeling after repeated allergen challenge detected noninvasively by MRI. Am J Physiol Lung Cell Mol Physiol 292, L644-L653.

17. Beckmann N., Cannet C., Karmouty-Quintana H., Tigani B., Zurbruegg S., Blé F.-X., Crémillieux Y. and Trifilieff A. (2007). Lung MRI for experimental drug research. European Journal of Radiology 64, 381-396.

18. B. Tigani, C. Cannet, H. Karmouty Quintana, F.-X. Blé, S. Zurbruegg, E. Schaeublin, J.R. Fozard, and N. Beckmann. Lung inflammation and vascular remodeling after repeated allergen challenge detected noninvasively by MRI. . Am. J. Physiol. Lung Cell. Mol. Physiol. 292:L644-L653 (2007).

19. V. Stupar, E. Canet-Soulas, S. Gaillard, H. Alsaid, N. Beckmann, and Y. Crémillieux. Retrospective Cine 3He ventilation imaging under spontaneous breathing conditions: a non-invasive protocol for small animal lung function imaging. NMR Biomed. 20:104-112 (2007).

20. N. Beckmann, R. Kneuer, H.U. Gremlich, H. Karmouty-Quintana, F.X. Blé, and M. Müller.In vivo mouse imaging and spectroscopy in drug discovery. NMR Biomed. 20:154-185 (2007).

21. H. Karmouty Quintana, C. Cannet, S. Zurbruegg, F.-X. Blé, L. Mazzoni, J.R. Fozard, and N. Beckmann.Capsaicin-Induced Mucus Secretion in the Airways Detected In Vivo and Non-Invasively by Magnetic Resonance Imaging (MRI). Br. J. Pharmacol. 150:1022-1030 (2007).

22. H. Karmouty Quintana, C. Cannet, S. Zurbruegg, F.-X. Blé, J. R. Fozard, C. P. Page, and N. Beckmann.Bleomycin-induced lung injury assessed non-invasively and in spontaneously breathing rats by proton MRI. J. Magn. Reson. Imaging 26:941-949 (2007).

23. N. Beckmann, C. Cannet, H. Karmouty-Quintana, B. Tigani, S. Zurbruegg, F.X. Blé, Y. Crémillieux, and A. Trifilieff. Lung MRI for experimental drug research. Eur. J. Radiol. 64:381-396 (2007).

24. F.-X. Blé, C. Cannet, S. Zurbruegg, H. Karmouty-Quintana, N. Frossard, A. Trifilieff, and N. Beckmann.Allergen-induced lung inflammation in actively sensitized mice assessed by MRI. Radiology, accepted for publication.

25. In vivo pharmacological evaluation of compound 48/80- induced airways oedema by MRI. H. Karmouty-Quintana, F.-X. Blé, C. Cannet, S. Zurbruegg, J.R. Fozard, C.P. Page, and N. Beckmann. Br. J. Pharmacol. 154:1063-72 (2008).

26. Allergen-induced lung inflammation in actively sensitized mice assessed with MR imaging. Blé FX, Cannet C, Zurbruegg S, Karmouty-Quintana H, Bergmann R, Frossard N, Trifilieff A, Beckmann N. Radiology 248:834-843 (2008).

27. Magnetic resonance methods and applications in pharmaceutical research. Rodríguez I, Pérez-Rial S, González-Jimenez J, Pérez-Sánchez J, Herranz F, Beckmann N, Ruíz-Cabello J. J. Pharm. Sci. 97:3637-65 (2008).

28. In Vivo Mouse Imaging and Spectroscopy in Drug Discovery (Beckmann N, Cannet C, Rausch M, Kneuer R, and Gremlich HU). In: „Imaging in CNS Drug Discovery and Development: Implications for Disease and Therapy“ (Editors: D. Borsook, L. Beccera, E. Bullmore, R. Hargreaves), pp. 229-250. Springer, New York (2009).

29. Capillary cerebral amyloid angiopathy is associated with vessel occlusion and cerebral blood flow disturbances. Thal DR, Capetillo-Zarate E, Larionov S, Staufenbiel M, Zurbruegg S, Beckmann N. Neurobiol Aging 30, 1936-1948 (2009).

30. Activation of the lung S1P1 receptor reduces allergen-induced plasma leakage in mouse (F.-X. Blé, C. Cannet, S. Zurbruegg, C. Gérard, N. Frossard, N. Beckmann, and A. Trifilieff). Br. J. Pharmacol. 158, 1295-1301 (2009).

31. In vivo assessments of mucus dynamics in the lungs using a Gd-Cy5.5-bilabeled contrast agent (F.-X. Blé, P. Schmidt, R. Kneuer, C. Cannet, C. Gérard, H. Karmouty-Quintana, S. Zurbruegg, K. Coote, H. Danahay, H.-U. Gremlich, and N. Beckmann). Magn. Reson. Med. 62, 1164-1174 (2009).

32. In Vivo Visualization of Macrophage Infiltration and Activity in Inflammation Using MRI (N. Beckmann, C. Cannet, A.L. Babin, F.-X. Blé, S. Zurbruegg, R. Kneuer, and V. Dousset). Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1, 272-298 (2009).

33. Spatially resolved assessment of serotonin-induced bronchoconstrictive responses in the rat lung using 3He ventilation MRI under spontaneous breathing conditions (K. Mosbah, V. Stupar, Y. Berthezène, N. Beckmann, and Y. Crémillieux). Magn. Reson. Med.,63:1669–1674 (2010).

34. Optical and Magnetic Resonance Imaging as Complementary Modalities in Drug Discovery. (B. S. Sandanaraj, R. Kneuer, and N. Beckmann). Fut. Med. Chem., 2, 317-337 (2010).

35. ENaC-mediated effects assessed by magnetic resonance imaging in a rat model of hypertonic saline-induced airways hydration (F.-X. Blé, C. Cannet, S. Collingwood, H. Danahay, and N. Beckmann). Br. J. Pharmacol., 160, 1008–1015 (2010).

36. - Non-Invasive Small Rodent Imaging: Significance for the 3R Principles. Beckmann N, Maier P. In: „Small Animal Imaging, Basics and Practical Guide“ (Editors: Kiessling F, Pichler BJ, Hauff P), pp. 47-57. Springer, Heidelberg (2011).

37. Non-Invasive Assessment of Bleomycin-Induced Lung Injury and the Effects of Short Term Glucocorticosteroid Treatment in Rats Using MRI. Babin AL, Cannet C, Gérard C, Wyss D, Page CP, Beckmann N. J Magn Reson Imaging, in press (2010).
Reson Med 63, 1669-1674 (2010).

38. A View on Imaging in Drug Research and Development for Respiratory Diseases. van Echteld CJA, Beckmann N. J Pharmacol Exp Ther, in press (2010).

39 Bleomycin-Induced Lung Injury in Mice Investigated by MRI: Model Assessment for Target Analysis. Babin AL, Cannet C, Gérard C, Saint-Mezard P, Page CP, Sparrer H, Matsuguchi T, Beckmann N. Magn Reson Med, submitted (2011).