**3. MRI cardiac imaging: The current status and future perspectives**

Mouse cardiac MRI emerged as a logical consequence to the human and mouse genome mapping initiatives and parallel developments and advances in human cardiac MRI in the late 1980's and early 1990's, focusing on establishing MRI as the 'one-stop-shop' in clinical practice. Correspondingly, technological advances and developments of novel hardware and pulse sequences were fairly limited, merely revolving on the scalability of existing technology to match smaller field-of-view acquisitions for mouse and rat imaging. Manning et al. [Manning 1990] and Shapiro [Shapiro 1994] first reported cardiac MRI-based LV mass estimations, and Siri et al. [Siri 1997] the first mouse cardiac imaging results from a 9.4 T system. Subsequent to such early studies were the first quantitative studies of function [Ruff 1998] and myocardial mass in diseased mice post-hypertrophy induction using the βadrenergic agonist isoproterenol (ISO) [Slawson 1998]. Despite attempts early on to utilize conventional 1.5T clinical systems to conduct mouse cardiac MRI [Franco 1998], it was soon realized that dedicated, high field, high resolution mouse systems were necessary for imaging, a realization that drove technological evolution and migration to field strengths of 4.7-11.7 T systems [Wiesmann 1998].

Collectively, efforts over the last 14 years targeted the study of global [Ruff 1998, Wiesmann 1998, Wiesmann 2002, Schneider 2003, Zhou 2003] and regional cardiac function [Epstein 2002, Zhou 2003, Heijman 2004, Zhong 2010] in transgenic and wildtype-littermate control mice as a basis for image-based phenotyping [Wiesmann 2000, Wiesmann\_Card\_Magn\_Reson 2001, Chuang 2010]. They also addressed models of disease (ischemia-infarction-reperfusion [Nahrendorf 2000, Ross 2002] and heart failure [Wiesmann 2002, Schneider 2003, Schneider 2004]). During the evolution of such prior attempts, noted advances in the mouse physiological status and its maintenance in high-field horizontal or vertical systems [Schneider 2003, Schneider 2004, Frydrychowicz 2007], and electrocardiographic (ECG) and respiratory gating strategies have been reported [Cassidy 2004, Bishop 2006, Hiba 2007, Heijman 2008, Bovens 2011] over the years. Other prominent work on mouse MRI also includes cardiac studies in imaging-based embryogenesis and development [Johnson 2002, Wiesmann 2000], perfusion [Kober 2005, Streif 2005], metabolism [Chacko 2000, Weiss 2002], coronary artery imaging [Ruff 2000], and stress testing [Williams 2001].

Only recently have efforts been completed to quantify the murine motional patterns and regional cardiac mechanical function [Zhou 2003, Streif 2005, Zhong 2010] towards the study of function and dysfunction using advanced techniques, including black-blood CINE [Berr 2005], spin-tagging [Epstein 2002, Liu 2006], DENSE [Zhong 2010], and HARP [Osman 1999, Kuijer 2001]. This section discusses some of the fundamental aspects of mouse preparation, positioning, physiology and its maintenance during MRI, advances in hardware and pulse sequence acquisitions, image processing techniques, and global and regional cardiac phenotyping, as these are complemented with recent findings from our group.

#### **3.1. Mouse physiology - Maintenance and stability during MRI**

354 Practical Applications in Biomedical Engineering

function exists, compared to the conscious state.

4.7-11.7 T systems [Wiesmann 1998].

imaging [Ruff 2000], and stress testing [Williams 2001].

and 75–50% N2O. However, despite the optimization of murine physiological conditions under anesthesia, it is yet not possible from this study to determine whether the mechanism of action involves transient sympathetic activation, steroid release, a direct effect of ISO, or a combination of such effects. The overall effects of ISO may be the result of opposing vasodilatory and vasoconstrictive effects either directly (vasodilation) or secondary to an anesthesia-induced decrease of the heart's metabolic demand. Surely, the major limitation of all studies under anesthesia is that a noted cardio-depressive effect on basic cardiovascular

**3. MRI cardiac imaging: The current status and future perspectives** 

Mouse cardiac MRI emerged as a logical consequence to the human and mouse genome mapping initiatives and parallel developments and advances in human cardiac MRI in the late 1980's and early 1990's, focusing on establishing MRI as the 'one-stop-shop' in clinical practice. Correspondingly, technological advances and developments of novel hardware and pulse sequences were fairly limited, merely revolving on the scalability of existing technology to match smaller field-of-view acquisitions for mouse and rat imaging. Manning et al. [Manning 1990] and Shapiro [Shapiro 1994] first reported cardiac MRI-based LV mass estimations, and Siri et al. [Siri 1997] the first mouse cardiac imaging results from a 9.4 T system. Subsequent to such early studies were the first quantitative studies of function [Ruff 1998] and myocardial mass in diseased mice post-hypertrophy induction using the βadrenergic agonist isoproterenol (ISO) [Slawson 1998]. Despite attempts early on to utilize conventional 1.5T clinical systems to conduct mouse cardiac MRI [Franco 1998], it was soon realized that dedicated, high field, high resolution mouse systems were necessary for imaging, a realization that drove technological evolution and migration to field strengths of

Collectively, efforts over the last 14 years targeted the study of global [Ruff 1998, Wiesmann 1998, Wiesmann 2002, Schneider 2003, Zhou 2003] and regional cardiac function [Epstein 2002, Zhou 2003, Heijman 2004, Zhong 2010] in transgenic and wildtype-littermate control mice as a basis for image-based phenotyping [Wiesmann 2000, Wiesmann\_Card\_Magn\_Reson 2001, Chuang 2010]. They also addressed models of disease (ischemia-infarction-reperfusion [Nahrendorf 2000, Ross 2002] and heart failure [Wiesmann 2002, Schneider 2003, Schneider 2004]). During the evolution of such prior attempts, noted advances in the mouse physiological status and its maintenance in high-field horizontal or vertical systems [Schneider 2003, Schneider 2004, Frydrychowicz 2007], and electrocardiographic (ECG) and respiratory gating strategies have been reported [Cassidy 2004, Bishop 2006, Hiba 2007, Heijman 2008, Bovens 2011] over the years. Other prominent work on mouse MRI also includes cardiac studies in imaging-based embryogenesis and development [Johnson 2002, Wiesmann 2000], perfusion [Kober 2005, Streif 2005], metabolism [Chacko 2000, Weiss 2002], coronary artery

Only recently have efforts been completed to quantify the murine motional patterns and regional cardiac mechanical function [Zhou 2003, Streif 2005, Zhong 2010] towards the study Even if physiological protocols are easier to optimize on the bench, murine cardiac MRI imposes additional stringent challenges that relate to the animal's heart rate, pressure monitoring, and thermoregulation [Schneider 2004, Hedlund 2008, Constantinides\_ILAR 2011]. While most research sites are equipped with commercial mouse imaging systems, a number of adjustments often need to be implemented to ensure proper physiological maintenance (including, but not limited to, computer controlled ECG, breathing, temperature monitoring systems and MR-compatible [fiber-optic and other] devices) [Figure 6]. Often specially designed negative-feedback air-flow systems are interfaced with closedbore scanner systems to facilitate fast and efficient bore air-heating, compensating for the lower bore temperatures due to the cryogenic environment of the magnet and gradient coils.

Proper mouse positioning almost always requires specially designed cradles to fit imaging probes (often constructed on specially designed casings that fit in fast-switching, high-slew rate gradient inserts) and scanner high-performance RF coil and gradient inserts.

Mouse cradle designs have evolved in complexity over the past 14 years and include those custom-made and commercially available types. Most of them are customized to allow mouse placement in prone and supine positions, placement of non-magnetic metallic or carbon-fiber ECG electrodes on the front paws and limbs, a rectal probe and an inflated air-bellow, for temperature and respiration monitoring [Figure 6]. Also of importance is the administration of anesthesia gases, often accomplished via a nose cone (with mice being obligatory nostril breathers), with tubing that connects to a vaporized-flowmeter device system (freely breathing [Schwartz 2000]), or directly to a ventilator device (achieving time-synchronous ventilation) as reported previously [Gilson 2005, Constantinides\_ILAR 2011]. Special arterial or venous lines (tail vein, carotid vein or artery) are often adapted to allow pharmacologic infusions or arterial/ventricular blood pressure monitoring [Constantinides\_ABME 2011]. Intraperitoneal injections are also preferred for anesthesia or contrast agent infusions.

Maintaining the mouse under optimal physiological conditions for a 60-90 minute MR imaging study, is a formidable and challenging task. Often, the HR value may be misleading as an indicative biomarker of proper physiological status (masking an underlying hypotension or cardiac contractile down-regulation [Constantinides\_ILAR 2011]), especially after prolonged anesthesia exposure, excessive infusion of electrolytes (for blood volume maintenance), or following pharmacologic challenges. Therefore, care must be exercised in the initial induction, proper inhalational anesthesia administration, proper acclimatization of the animal to its imaging environment, and careful control and monitoring of its temperature and

other critical physiological parameters (including, but not limited to HR, coefficient of variation [CV], mean arterial pressure [MAP], blood pH, glucose, and insulin) maintaining homeostasis throughout the study as shown in Table 1 [Constantinides\_ILAR 2011].

Study of the Murine Cardiac Mechanical Function Using Magnetic Resonance Imaging:

**tRR**

114- 134ms (N=4, M)

108- 150ms (N=3-6, M)

114- 124ms (N=3-4, M)

13±3 (N=7,M)

8±2 (N=7,M)

33±2 (N=7,M)

25±6 (N=7,M)

6±2 (N=6,M)

10±4 (N=5,M)

6±3 (N=5,M)

10±3 (N=5,M)

**Anesthesia/Inhalati on Mixture** 

> O2=100 %

> O2=100 %

> O2=100 %

% O2 =75% 10±2

% O2 =20% 8±1

O2 =50% N2O=50 %

O2 =75% N2O=25 %

*3.2.2. Gradient coil technology* 

computerized cradle positioning systems.

ISO=1.0 %

ISO=1.5 %

ISO=2.0 %

ISO=1.5

ISO=1.5

ISO=1.5

ISO=1.5 %

ISO=1.5 %

% O2 =50%

**HR [bpm] CVHR**

478±11.4 (N=7,M)

475±21.6 (N=6, M)

490±15.0 (N=5, M)

516±8.7 (N=5, M)

14±2 (N=7, M)

11±1 (N=7, M)

14±3 (N=7, M)

(N=7, M)

9±1 (N=6, M)

(N=5, M)

10±2 (N=5, M)

7±2 (N=5, M)

**MAP [mmHg] CVMAP**

92±2 (N=7, M)

90±13 (N=6, M)

93±3 (N=5, M)

94±3 (N=5, M)

the C57BL/6 Mouse, published in ILAR e-Journal 52(2), available online at *http://dels-*

*old.nas.edu/ilar\_n/ilarjournal/52\_2/PDFs/v5202e-Constantinides.pdf*].

**Table 1.** Summary of various cardiovascular and biochemical physiological indices of anesthetized mice using Isoflurane from bench studies. Stable mouse responses were obtained under both bench and scanner experimental conditions for periods spanning 1-1.5 hrs post-induction. [This table originally appeared in Constantinides C et al., Effects of Isoflurane Anesthesia on the Cardiovascular Function of

The requirements for increased spatial resolution acquisitions (of orders matching cellular size of approximately 100-200 μm3) led to fast switching, high-amplitude (up to 1000 mT/m), high-slew rate (up to 11250 mT/s) gradients (exhibiting linearity of better than ±3-5%/mm) for ex-vivo constructions of atlases [Johnson 2002] or in-vivo high-resolution isotropic cardiac imaging [Bucholz 2008, Perperidis 2011]. Often associated with extra inserts (of approximately 6-20 cm in inner diameter), such gradients impose further spatial restrictions in animal placement and monitoring during cardiac imaging. However modern, commercially available systems are often equipped with actuator-controlled manual or

The Current Status, Challenges, and Future Perspectives 357

**[mg/ml]** 

176.3±26.1 - 188.8±11.1 (N=4/5, M)

139.4±30.1 -192±11.9 (N=4, M)

**Insulin [ng/ml]** 

3.4±0.5- 4.2±0.9 (N=4/5, M)

3.54±0.3- 4.24±0.6 (N=4, M)

**[ms] pH Glucose** 

7.3±0.1- 7.4±0.1 (N=9/5, M)

7.3±0.1- 7.3±0.2 (N=9/6, M)

**Figure 6.** Physiological monitoring of mice during MRI using a dedicated recording system and the Labview software; (b) specially designed mouse positioning cradle, and (c) RF coil-mouse cradle assembly for a small-bore, high field animal scanner.[Photographs acquired from the Center for In Vivo Microscopy at Duke University Medical Center, an NIH/NIBIB National Biomedical Technology Resource Center].
