*2.2.1. Mechanisms of anesthesia action*

Linus Pauling [Pauling 1952] was one of the first scientists to attempt to explain the molecular and cellular mechanisms of action of anesthetics. Nevertheless, his proposed theory on clathrate formation post-anesthesia administration, proved inaccurate. With recent advances in molecular biology, new published evidence justifies the prominent role of the cellular chlorine channel as a mediator for anesthesia induction and maintenance [Brunson 2008, Maze 2008]. Specifically, hyperpolarizing chlorine channel currents lead to inhibition of cellular excitability and hypnotic action. It appears that one of the major hypnotic action centers is the locus coeruleus in the central nervous system, with mediatory action referred to the α2-adrenergic receptors via cyclic-AMP-mediated transduction pathways.

At the integrative physiological level, the effects of anesthesia target multiple cellular sites (Figure 3) and thereby often lead to cardio-depression. Major effects include their potency in inducing vasodilation of both the cerebral and coronary vasculature [Toyama 2004] leading to increased perfusion. A rapid hyperglycemic effect is often expressed (primarily via the sympathetic nervous system innervating the liver) causing immediate hormonal (catecholamine) release from the adrenal medulla [Durand 2009]. Glucose metabolic rates are also down-regulated (via inhibition of ATP synthesis in mitochondria), eventually leading to impairment of glucose tolerance (mediated via enzymatic protein activity in the liver). Also observed is an eventual oxidative phosphorylation decoupling.

#### *2.2.2. Types of anesthesia*

Widely used anesthetics for animal research are categorized into injectable (such as Ketamine/Xylazine, propofol, lidocaine, nembutal) and inhalational (such as isoflurane, halothane, and sevoflurane). Hedlund et al. [Hedlund 2008] and Brunson et al. [Brunson 2008] have published excellent summaries of the different types of anesthetics used for rodents to which the reader is referred.

#### *2.2.3. Basal physiology and maintenance*

While anesthesia usage causes physiological changes [Hildebrandt 2008], proper protocols and administration methodologies (type, dose) can achieve optimal conditions of animal study, maintain animal stability and homeostasis, and minimize time-induced accumulated Study of the Murine Cardiac Mechanical Function Using Magnetic Resonance Imaging: The Current Status, Challenges, and Future Perspectives 351

350 Practical Applications in Biomedical Engineering

through myocardial oxygen consumption changes.

*2.2.1. Mechanisms of anesthesia action* 

pathways.

*2.2.2. Types of anesthesia* 

rodents to which the reader is referred.

*2.2.3. Basal physiology and maintenance* 

binding sensitivity of the contractile proteins to calcium, on conduction and excitability, and possibly on other sarcoplasmic reticular sites [Price 1980]. Also prominent are effects on the central and peripheral nervous system, but most notable are effects on the metabolism (through mitochondrial vasomotor changes in coronary circulation and perfusion, vasodilation, and blood flow changes in the microvasculature [Kober 2005], possibly synergistic to Nitric Oxide), and the decoupling of oxidative phosphorylation manifested

Linus Pauling [Pauling 1952] was one of the first scientists to attempt to explain the molecular and cellular mechanisms of action of anesthetics. Nevertheless, his proposed theory on clathrate formation post-anesthesia administration, proved inaccurate. With recent advances in molecular biology, new published evidence justifies the prominent role of the cellular chlorine channel as a mediator for anesthesia induction and maintenance [Brunson 2008, Maze 2008]. Specifically, hyperpolarizing chlorine channel currents lead to inhibition of cellular excitability and hypnotic action. It appears that one of the major hypnotic action centers is the locus coeruleus in the central nervous system, with mediatory action referred to the α2-adrenergic receptors via cyclic-AMP-mediated transduction

At the integrative physiological level, the effects of anesthesia target multiple cellular sites (Figure 3) and thereby often lead to cardio-depression. Major effects include their potency in inducing vasodilation of both the cerebral and coronary vasculature [Toyama 2004] leading to increased perfusion. A rapid hyperglycemic effect is often expressed (primarily via the sympathetic nervous system innervating the liver) causing immediate hormonal (catecholamine) release from the adrenal medulla [Durand 2009]. Glucose metabolic rates are also down-regulated (via inhibition of ATP synthesis in mitochondria), eventually leading to impairment of glucose tolerance (mediated via enzymatic protein activity in the

Widely used anesthetics for animal research are categorized into injectable (such as Ketamine/Xylazine, propofol, lidocaine, nembutal) and inhalational (such as isoflurane, halothane, and sevoflurane). Hedlund et al. [Hedlund 2008] and Brunson et al. [Brunson 2008] have published excellent summaries of the different types of anesthetics used for

While anesthesia usage causes physiological changes [Hildebrandt 2008], proper protocols and administration methodologies (type, dose) can achieve optimal conditions of animal study, maintain animal stability and homeostasis, and minimize time-induced accumulated

liver). Also observed is an eventual oxidative phosphorylation decoupling.

**Figure 3.** Schematic representation of a typical myocyte with indicative (highlighted) target sites of inhalational anesthesia.

effects, especially for prolonged imaging studies often exceeding 45 minutes in duration. Apart from physiological indices of hormonal release, respiration and metabolism, carefully controlled cardiac indices (such as heart rate, ejection fraction, arterial pressure, and heart variability) ensure proper conditions of study of the cardiovascular system avoiding detrimental hypotension-induced blood volume changes, metabolic and contractile downregulation, and arrhythmogenicity. Heart variability (HRV) analyses have also been applied for phenotypic screening of transgenic mice, study of heart rhythm mediators through signaling pathways as well as the effects of pharmacologic intervention on intrinsic heart rhythm and arrythmogenesis [Thireau 1997, Bernston 1997, Gehrmann 2000]. Standardization of HRV analyses tools for mice have, however, been limited due to the numerous data acquisition types and analysis techniques employed. Most importantly, variability of HRV is dependent on the type of anesthesia being used, that may also be further influenced by anesthetic balancing agents such as nitrous oxide (N2O), medical air, and oxygen. Interpretation of HRV results has also been difficult [Hoit 2004], due to their dependence on a number of factors including aging, posture, circadian variability, and the duration of the sampling periods used, with recent evidence supporting the fact that the primary contribution of autonomic activity in the mouse is due to the sympathetic tone, in contrast to the parasympathetic vagus contribution [Janssen 2002, Janssen 2000, Constantinides\_IEEE 2010].

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

Despite such previous work, the extent of the contribution of sympathetic and parasympathetic tone to heart rate (HR) and HRV in the mouse is still a matter of debate. Recent, carefully controlled studies based on the level of anesthesia and additional factors that relate to animal preparation indicate that overall, HRV is expected to be generally lower during anesthesia in comparison to the conscious state. Indicative in HRV is an SDNN reduction under FiO2 conditions at 50% but also a noted increase in the standard deviation of averages of all normal R-R intervals (SDNN) and the square root of mean squared differences between adjacent normal R-R intervals (RMSSD) as a result of N2O administration. Variation of FiO2 seems to result in prominent effects only at specific levels (50, 100%). Since HRV indices are determined by an inter-play between both mechanical and

neural factors, the exact mechanisms responsible for this effect are still unknown.

**RMSSD [ms]**

**SDNN [ms]**

**Figure 5.** Heart rate variability indices with varying N2O/O2 values (25-75% and 50-50%) at different time intervals post-anesthesia induction. (Top) SDNN and (bottom) RMSSD variation (n=3-6 per group).

[0-15] [15-30] [30-45] [45-60] [60-75] **Time Interval [min]**

[0-15] [15-30] [30-45] [45-60] [60-75] **Time Interval [min]**

Overall, the major practical benefit is that the protocol described for physiological studies of mice under anesthesia has the potential for high reproducibility in diagnostic modalities including MRI, microCT, ultrasound, and microPET. Elicited results show that the optimal ISO anesthetic regimen for mice is a dose of approximately 1.5% v/v mixed with 25–50% O<sup>2</sup>

[Reproduced from Constantinides et al. [Constantinides\_IEEE 2010] with IEEE permission].

The Current Status, Challenges, and Future Perspectives 353

N2O=25% N2O=50%

N2O=25% N2O=50%

**Figure 4.** Heart rate variability indices at varying FiO2 values. (Top) HR, R-R intervals (NN), (bottom) SDNN, and RMSSD variation. [Reproduced from Constantinides C et al. [Constantinides\_IEEE 2010] with IEEE permission].

Despite such previous work, the extent of the contribution of sympathetic and parasympathetic tone to heart rate (HR) and HRV in the mouse is still a matter of debate. Recent, carefully controlled studies based on the level of anesthesia and additional factors that relate to animal preparation indicate that overall, HRV is expected to be generally lower during anesthesia in comparison to the conscious state. Indicative in HRV is an SDNN reduction under FiO2 conditions at 50% but also a noted increase in the standard deviation of averages of all normal R-R intervals (SDNN) and the square root of mean squared differences between adjacent normal R-R intervals (RMSSD) as a result of N2O administration. Variation of FiO2 seems to result in prominent effects only at specific levels (50, 100%). Since HRV indices are determined by an inter-play between both mechanical and neural factors, the exact mechanisms responsible for this effect are still unknown.

352 Practical Applications in Biomedical Engineering

**RMSSD [ms]**

**SDNN [ms]**

**NN Interval [ms]**

FIO=100 FIO2=75 FIO2=50 FIO2=20

**HR [bpm]**

[0-15] [15-30] [30-45] [45-60] [60-75] **Time Interval (min)**

> FIO2=100 FIO2=75 FIO2=50 FIO2=20

FIO2=100 FIO2=75 FIO2=50 FIO2=20

FIO2=100 FIO2=75 FIO2=50 FIO2=20

[0-15] [15-30] [30-45] [45-60] [60-75] **Time Interval (min)**

[0-15] [15-30] [30-45] [45-60] [60-75] **Time Interval [min]**

with IEEE permission].

**Figure 4.** Heart rate variability indices at varying FiO2 values. (Top) HR, R-R intervals (NN), (bottom) SDNN, and RMSSD variation. [Reproduced from Constantinides C et al. [Constantinides\_IEEE 2010]

[0-15] [15-30] [30-45] [45-60] [60-75] **Time Interval [min]**

**Figure 5.** Heart rate variability indices with varying N2O/O2 values (25-75% and 50-50%) at different time intervals post-anesthesia induction. (Top) SDNN and (bottom) RMSSD variation (n=3-6 per group). [Reproduced from Constantinides et al. [Constantinides\_IEEE 2010] with IEEE permission].

Overall, the major practical benefit is that the protocol described for physiological studies of mice under anesthesia has the potential for high reproducibility in diagnostic modalities including MRI, microCT, ultrasound, and microPET. Elicited results show that the optimal ISO anesthetic regimen for mice is a dose of approximately 1.5% v/v mixed with 25–50% O<sup>2</sup>

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 function exists, compared to the conscious state.

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

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

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

phenotyping, as these are complemented with recent findings from our group.

rate gradient inserts) and scanner high-performance RF coil and gradient inserts.

injections are also preferred for anesthesia or contrast agent infusions.

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

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

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

The Current Status, Challenges, and Future Perspectives 355
