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Introduction

Animal models have long played an important role in pharmaceutical research, and their use is well embedded in many critical steps in the drug discovery process. The highly structured toxicology testing regimens and measurements of absorption, distribution, metabolism and excretion (ADME) in multiple animal species are required by regulatory agencies for approval of an experimental drug candidate to be administered to humans. Drug discovery also utilizes a wide range of animal studies geared towards risk management of expensive clinical trials by improving and predicting the probability of success in the clinic. Examples of the latter include target validation, lead optimization, and development of biomarkers of disease, compound efficacy and toxicology; some biomarkers can readily be used in the clinic allowing response in the human to be tested with the same metrics used in preclinical development, with minimal or no change in experimental design. The sequencing of human and mouse genome has opened yet another rich area of research using genetically engineered mice with new models of disease for studying disease progression and response to therapy, for providing proof of biology, and providing the possibility of new tests for the safety assessment of compounds (Mouse Genome Sequencing Consortium 2002; The International Human Genome Mapping Consortium 2001).

Imaging offers several advantages over traditional research readouts in animals. While noninvasive imaging of live animals allows longitudinal monitoring, and can significantly reduce the number of animals needed to address a biological question, imaging readouts are also believed to be more closely related to the disease phenotype, thus providing a more direct correlation between therapeutic effect and the measurement (Lawrence & Mackey 2008; Rudin 2008; Wang & Yan 2008; Badea et al. 2008; Henkelman et al. 2005; Beckmann & Rudin 2006). This is especially true when the disease phenotype has a spatial characteristic. No single imaging modality will answer all possible questions, with each modality’s strengths lying in different domains with respect to the type of information it provides. A full discussion of the use of each imaging modality in drug discovery is beyond the scope of this chapter and the reader is referred to many excellent reviews in the literature (Beckmann & Rudin 2006; Hargreaves 2008; Gwyther & Schwartz 2008; Ripoll et al. 2008). This chapter will focus on Functional Magnetic Resonance Imaging (fMRI) with selected examples to illustrate its application to drug discovery using animal models.

The term fMRI is used here in a broad sense, representing all MRI procedures that provide information about brain function. In this sense, the term refers to techniques such as the now popular BOLD (Ogawa et al. 1990) or cerebral blood volume mapping (CBV) functional MRI techniques (Kennan et al. 1998), which measure regional hemodynamic phenomena associated with neuronal activation, in response to external stimuli or task execution, or Arterial Spin Labeling (ASL) techniques (Williams et al. 1992), which can be used to quantitatively measure regional Cerebral Blood Flow (rCBF) both at rest, and in response to external stimuli.

Exploiting the full potential of fMRI in animal studies requires special consideration. While controlling motion of the subject is important in any MRI experiment, fMRI places stricter standards because of very small signal changes observed, and the need for the signals to be quantified. Animal motion can be controlled either through anesthesia or using a neuromuscular blocker as a muscle relaxant, or in conscious animals, with a combination of restraint and animal acclimation to scanning conditions. While fMRI applications, particularly in rodents have predominantly been carried out on anesthetized animals, the value of conscious animal studies for functional readouts is increasingly being recognized (Lahti et al. 1998; Borsook et al. 2007). Even when the animal is completely immobilized, occasionally, physiological motion related to cardiac or respiratory processes needs to be accounted for; and their effects may be minimized by synchronization of signal acquisition to the motion’s periodicity (Zhao et al. 2009), or by post-processing of data (Brooks et al. 2008).

The quality of fMRI signals, particularly with regard to sensitivity, spatial representation, and temporal stability are influenced by the MR measurement technique and other instrumental factors, and the scanning protocol needs to be tailored so as to optimize the desired output characteristics. Furthermore, robustness of the fMRI signal needs to be demonstrated through test-retest data so that studies can be designed with adequate statistical power (Zhao et al. 2008a).

In all fMRI experiments, it is essential to ensure that the signals measured are the direct result of the specific biological function being studied, and not due to any unrelated physiological changes; therefore maintaining a stable state of physiology represents a significant effort in animal fMRI. Key physiological parameters such as heart rate, arterial blood pressure, body temperature and blood gases are usually monitored throughout the experiment. Careful attention needs to be given to the choice of anesthetic, or any possible influence of restraint, such as stress to ensure that these factors do not interfere with the biological function being measured or the measurement itself.

To answer specific questions relating to CNS, a measure of neuronal function is desired, and one of the most challenging tasks is to confirm that the fMRI signal or signal change observed is a true reflection of the neuronal function being interrogated. This is because fMRI does not directly measure neuronal activity. Rather, it provides either a measure of hemodynamic change in response to this neuronal activity, or a resting physiological measure such as CBF or CBV, that is reflective of steady state neuronal activity. Confirming that the fMRI signal change is a direct result of neuronal activity is non trivial; validity of this hypothesis is often sought through additional experiments involving pharmacological tools, or by matching the spatial and temporal characteristics of fMRI data with those that provide direct information on neuronal activity, e.g., electrophysiological recordings (Logothetis et al. 2001; Shmuel et al. 2006). Supporting experiments may involve the use of pharmacological agents known to modulate the neuronal activity either by acting directly on the target neurons (at the synaptic level) or at some point upstream or downstream in a cascade of interconnected events.

The neural activity-induced hemodynamic changes can be measured by fMRI as a BOLD effect, representing the net change in deoxyhemoglobin due to cerebral metabolic rate of oxygen (CMRO2) and CBF, as a change in cerebral blood volume (denoted CBV for simplicity, but includes brain and spinal cord), or as a pure CBF change. The most appropriate choice of fMRI readout will depend on several factors. For example, BOLD is the simplest to implement and is the most widely used, while blood volume measurements with a blood volume contrast agent provide enhanced sensitivity and better spatial specificity (Zhao et al. 2006). BOLD and CBV fMRI rely on a change in signal due to a stimulus, and requires measurement of response to an acute stimulus (i.e., in the same scanning session), and is not suitable for monitoring resting hemodynamic effects reflective of disease or therapy. Measurement of CBF using a quantitative technique such as ASL allows measuring resting blood flow as a marker of neuronal activity, but is also amenable to measuring stimulus-induced changes. In humans, while BOLD and ASL are commonly used, the need for an exogenous contrast agent makes CBV fMRI less common.

Despite the challenges, fMRI in animals can provide a wealth of valuable information often not obtainable by other modalities. It allows adequate flexibility to tailor experiments to obtain information critical to drug discovery process, such as target validation, lead optimization, dose selection, and to provide proof of biology and mechanism of action for novel agents.

Technical Approaches to Animal fMRI

Due to the sensitivity of the fMRI techniques to subject motion, it is imperative that every precaution be taken to maintain subjects as immobile as possible. In any brain MRI experiment, motion artifacts can be greatly reduced by mechanical restraint of the head. In fMRI of conscious animals, anxiety and stress can significantly affect brain function (Takamatsu et al. 2003), and animals need to be acclimated to the restraint condition in order to minimize stress. The acclimation process typically consists of daily conditioning of the animals to simulations of the conditions observed by the animal while in the scanner, i.e., long periods of restraint and high noise level. It has been shown that through the acclimation process animal stress-related physiology and stress hormone levels gradually normalize and motion is reduced (Fig. 1, (Welsh et al. 2008a; King et al. 2005)). Even when physical movement of the animal is constrained by restraint or with the use of pharmacological agents, motion can also arise from physiological processes such as respiration and heart beat. Respiration can either directly induce motion in the brain or spinal cord, or cause signal instabilities as a result of periodic alterations in field homogeneity. In an fMRI study of the cervical spinal cord, it was demonstrated that respiration caused an MRI signal fluctuation large enough to override the fMRI signal change, completely obliterating any statistical significance of the stimulus-induced signal (Zhao et al. 2009). Synchronization of the data acquisition to the respiratory cycle (Zhao et al. 2009) and/or post-processing of the data to filter out the respiration-induced MRI signal fluctuation has allowed these motion artifacts to be removed effectively (Brooks et al. 2008).

Fig. 1
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Reproduced from King et al. 2005. Time evolution of stress-related physiological parameters, heart and respiratory rate, and stress hormone cortisol, indicating normalization of stress in acclimated rats with length of acclimation

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Very significant challenges posed in maintaining conscious animals completely still and devoid of stress, particularly during administration of stimuli, have resulted in the majority of animal fMRI studies being carried out predominantly under anesthesia. However, as anesthetics are pharmacological agents, their choice and use in drug development application need to be carefully considered because of the potential confound effects such as drug–drug interaction (Tsukada et al. 1999) physiological changes (CBF, CBV, and blood pressure, induced by the anesthetic) (Welsh et al. 2008a; Sicard et al. 2003). In studies of CNS function, knowledge of the effects of the anesthetic on brain metabolism and hemodynamic function (CBF, CBV), and of interactions between the anesthetic and the specific system under investigation needs to be considered. Some anesthetics are known to affect specific neuro-receptor systems and therefore should be avoided in studies tackling these respective systems. For example, ketamine, is a noncompetitive antagonist of NMDA receptors and also affects the dopamine system (Tsukada et al. 2000). Propofol, was shown to interfere with processing of vibrotactile information in humans (Bonhomme et al. 2001). Isoflurane has also been shown to affect the dopamine system (Tsukada et al. 1999) and vascular responsivity to CO2 challenge (Sicard et al. 2003) and the vasodilator Acetazolamide (Welsh et al. 2008a).

The ability to scan awake animals creates an opportunity to investigate neurologic processes under true physiologic conditions, and should provide more clinically relevant information during the drug development process. However, careful choice of anesthetic has allowed useful information to be obtained in several key experiments, and fMRI in anesthetized animals remains a viable platform for studying CNS function.

To date, most animal fMRI studies have been performed under anesthesia with α-chloralose or isoflurane (Lee et al. 1999; Masamoto et al. 2007). Alpha-chloralose is a chlorinated acetal derivative of glucose with anesthetic and sedative properties. Its advantages for fMRI studies are that it: (1) preserves metabolic coupling for somatosensory stimulation (Ueki et al. 1992), (2) provides good stability of baseline blood flow (Lindauber et al. 1993), and (3) preserves cerebrovascular reactivity (Bonvento et al. 1994). A drawback with α-chloralose is that its use is often limited to “terminal” experiments due to invasive intubation and catheterization, preventing its use in animal fMRI to investigate longitudinal changes in brain function. Isoflurane, however can be used as an anesthetic for “survival” experiments (Masamoto et al. 2007), but it causes hypotension and respiratory depression, and is a potent cerebral vasodilator, all of which lead to a hemodynamic response confounding the true fMRI signals (Sicard et al. 2003).

Medetomidine (domitor) offers a viable alternative for “survival” studies and an anesthesia protocol has been developed and validated recently (Weber et al. 2006; Zhao et al. 2008b). Medetomidine is an α2−adrenoreceptor agonist which can provide sedation and anxiolysis, analgesia and some muscle relaxation (Lukasik & Gillies 2003). It is generally used as an adjuvant to reduce anesthetic requirements to tracheal intubation and surgical stimuli (Bol et al. 1999). It was first used in rat fMRI as an independent anesthesia by Weber et al. (2006). Since it is administered subcutaneously requiring no catheterization, the animal can be maintained in a free breathing state requiring no intubation, allowing longitudinal (survival) fMRI studies. With medetomidine anesthesia, well-localized activations in the somatosensory pathway (contralateral primary and secondary somatosensory cortex (SI & SII), contralateral thalamus) have been detected (Zhao et al. 2008b). Also, the incidence of activations in animals with Domitor anesthesia in thalamus and SII are higher than those under α-chloralose anesthesia (Keilholz et al. 2004). To determine reproducibility of long term fMRI signals under Domitor anesthesia, fMRI activations induced by electrical stimulation of bilateral rat forepaws were measured at three time points 1 week apart (personal communication from authors – unpublished). Figure 2a shows activation maps in a coronal slice through S1 for a representative rat at three time points 1 week apart. Figure 2b shows the time courses for three animals at the three time points. Figure 2c shows the maximum fMRI response, averaged over three animals at the three time points, and Fig. 2d shows the same data after normalization, to the response on day 1. Excellent reproducibility was observed between the three longitudinal measurements. Most importantly, as indicated in (Zhao et al. 2008b), with the low dose suggested by Weber et al. (2006), rats are in “a sedation level adequate for reliable fMRI experiments” rather than in an anesthetized state, and robust functional connectivity maps were detected between large cortical regions in two hemispheres and in the caudate putamen (CPu) of two hemispheres, consistent with the results from awake humans (Zhao et al. 2008b).

Fig. 2
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Reproducibility of BOLD fMRI signals in rats under domitor-anesthesia. Rats (n = 3) were subjected to bilateral electrical stimulation and fMRI activity maps were obtained using an EPI based BOLD technique at 7T. The same animals were scanned three times, at day 1, day 7 and day 14. (a) fMRI activation maps from a coronal slice through S1 of brain from one rat scanned on three different days. The pattern of activation at the three different time points are quite similar. (b) Time courses of fMRI responses from an ROI covering S1 of three different rats at the three different times. The temporal patterns of fMRI responses for the same rat at the three different times are also similar. Small variations exist in the temporal patterns of different rats. Bars under the time courses are the stimulation periods. (c) Averaged fMRI responses in S1 over three rats at the three different times (mean ± standard deviation). Stability of fMRI signals was examined by one-way repeated measures ANOVA: F2,6 = 0.08, p = 0.92, indicating that the fMRI signals are stable over different measuring time. (d) Normalized fMRI responses (normalized to the response on day 1) at three different times (mean ± standard deviation). The variation of the average normalized fMRI signal change over different sessions is less than 4%

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Application of fMRI to CNS Drug Discovery

In the following sections, we illustrate through selected examples, the use of animal fMRI to address specific questions in CNS drug discovery. fMRI of pain is an area of active clinical research to understand how the CNS processes pain, to provide an objective measure of pain, and to evaluate the analgesic efficacy of experimental drug candidates. An fMRI assay to test the analgesic effect will involve measuring the pharmacological modulation of a noxious stimulation-induced activation in CNS, and should ideally have the following characteristics (Wise et al. 2002; Wise & Tracey 2006): (1) the stimulation-induced fMRI signal is repeatable in a time span of hours to acquire sufficient data for a baseline condition and during analgesic application; (2) the analgesics can be administered intravenously to achieve a fast onset; and (3) fMRI signals should have high sensitivity (high contrast to noise ratio) and a wide dynamic range. A pain fMRI assay that satisfies the criteria above has been developed in a rat model and validated with the known analgesic lidocaine as a positive control. The assay uses noxious electrical stimulation as a pain source. Subcutaneously applied noxious stimulus (electrical pulse with 2 ms width, 5 mA amplitude) has been proved to robustly evoke the C-fiber response based on electrophysiology studies (Le Bars et al. 1979) and it has been used as a surrogate of neuropathic pain in human studies (Klein et al. 2005). The reproducibility of such noxious electrical stimulation-induced fMRI signals has also been well established (Zhao et al. 2008a). To achieve a fast onset of the analgesia effect, an intravenous delivery of the therapeutic agent is used rather than oral or other delivery methods with a slower absorption. A fast onset makes it possible for pharmacological effect to be assessed with the data acquired in the same experimental session, so the additional between-session sources of variance (i.e., differences in animal physiology, variations in electrical stimulation efficiency due to needle positions, magnet field homogeneity and its effect on T2*, signal-to-noise ratios, etc.) can be avoided. The sensitivity of the fMRI signal is increased using a blood volume contrast agent (blood volume (BV)-weighted fMRI) rather than BOLD fMRI. A commonly used BV contrast agent is superparamagnetic iron oxide particles, which remain in the vasculature and produce a susceptibility gradient in surrounding tissue (Kennan et al. 1998). BV-weighted fMRI in the spinal cord using noxious electrical stimulation of rat hindpaws gave excellent sensitivity and reproducibility (Zhao et al. 2008a). In that study, reproducibility of the signals was rigorously tested by examining the correlation of the pixel-wise fMRI activation signals between odd and even runs (Zhao et al. 2008a). Confirmation that the detected fMRI activity in the spinal cord was due to neuronal activity elicited by the electrical stimulation was provided by the fact that fMRI signal activity was located in the dorsal horn ipsilateral to the stimulated paw in the L3–L5 spinal cord segments, matching synapse location of the somatic peripheral fibers. In the cross-sectional direction, the highest noxious stimulation-induced neuronal activations are located in the middle of the ipsilateral dorsal horn, which is in agreement with the data from electrophysiological techniques, 2DG autoradiography, and C-FOS expression (Le Bars et al. 1979; Menetrey et al. 1977; Porro & Cavazzuti 1993), lending further support to the fact that we are indeed detecting a hemodynamic change directly related to noxious stimulation-induced neuronal activity.

Testing this spinal cord fMRI technique as an assay for pain is now presented with the known analgesic Lidocaine as a benchmark. Lidocaine, a sodium channel blocker, is widely used as a local anesthetic. When it is injected locally, it blocks peripheral nerve transmission, thereby attenuating the neural activity transmitted to CNS. To determine if BV-weighted fMRI of spinal cord (Zhao et al. 2008a, 2009) with noxious electrical stimulation in domitor anesthetized-rats can serve as a pain assay, lidocaine was locally injected around the stimulating electrodes in one hindpaw, while the same amount of saline was similarly injected around the stimulating electrodes in the other hindpaw as a control (both paws were subjected to electrical stimulation simultaneously). Figure 3 shows the activation maps obtained before and after injection of saline and lidocaine, respectively. Before local injection of lidocaine and saline, fMRI signals (left column in Fig. 3) show similar patterns of activation. After local subcutaneous injection of lidocaine, fMRI signals were ablated within 2 h and slowly recovered with time (the bottom row in Fig. 3). However, the fMRI signals after saline injection were relatively constant (the top row in Fig. 3). These results indicate that the BV-weighted fMRI in spinal cord can detect the blockage effect of locally injected lidocaine on the transmission of neural activity in peripheral nerves, verifying that BV-weighted fMRI can be used as a pain assay to test the analgesia effect of analgesics.

Fig. 3
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BV-weighted fMRI demonstrating the effect of locally injected lidocaine in blocking electrical stimulation induced activity in the spinal cord in rat. Lidocaine was locally injected around the stimulating electrodes in one hindpaw, while the same amount of saline was similarly injected around the stimulating electrodes in the other hindpaw as a control (both paws were subjected to electrical stimulation simultaneously). (a) and (b) show activation maps obtained from two saggittal slices containing the respective dorsal horn before and after injection of saline and lidocaine. Negative ΔS/S changes (blue/violet as indicated by the color bar) detected in the BV-weighted fMRI indicate an increase in spinal blood volume. After local subcutaneous injection of lidocaine, fMRI signals in the slice ipsilateral to the paw injected with lidocaine were ablated within 2 h and slowly recovered with time (b). However, the fMRI signals after saline injection were relatively constant (a). The spinal cords were outlined by the green lines. R rostral, V ventral

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We now present two examples of the use of fMRI to provide proof of biology and mechanistic information. The first is on fMRI investigation of pharmacological modulation of the co-agonist glycine site of the NMDA receptor. When investigating the effect of new compounds on a specific receptor target system, a study design that includes different pharmaceutical challenges to perturb the system in a predictable way can provide additional support for hypotheses on mechanism of action. This particular site has gained recognition as a promising therapeutic target to enhance glutametergic and dopaminergic system function with no or limited excito-toxic side effects. Compounds that inhibit glycine uptake via blockade of glial and neuronal transporter GlyT1 have been shown to increase extracellular glycine levels in rodents, and have shown clinical efficacy in ameliorating schizophrenic symptoms (Lechner 2006). The pharmacodynamic effects of novel therapeutics that work through indirect mechanisms, such as inhibition of GlyT1, are difficult to assess in vivo, requiring invasive techniques and/or terminal studies with large numbers of animals. In this context, fMRI is of great value in that it represents a noninvasive method providing a physiologic biomarker for the pharmacodynamic activity of such compounds for proof of concept, dose selection, etc. Here we explore the use of ASL to assess dynamic cerebral perfusion following administration of compounds that modulate the NMDA receptor function (Welsh et al. 2008b).

Figure 4 shows dynamic cerebral blood flow (CBF) measurements during administration of various pharmacological agents known to perturb the NMDA system. Administration of the specific NMDA glycine site agonist d-serine, increased cerebral perfusion compared to baseline perfusion and vehicle controls (not shown). Blockade of the glycine site with a highly selective NMDA glycine site blocker reduced this perfusion response. In contrast, administration of two doses of the GlyT1 inhibitor produced a robust decrease in cerebral perfusion compared to baseline perfusion and vehicle controls. Prior blockade of the glycine site with a highly selective glycine site blocker attenuated this decrease. A structurally different GlyT1 inhibitor also produced a robust decrease in perfusion (data not shown) supporting that the CBF increase is connected with NMDA activity and not due to a systemic effect. Arterial CO2 remained constant for all animals during the course of scanning. Potential confounding covariates such as blood pressure were not measured during scanning, but separate bench experiments showed no increase in mean blood pressure after administration of the GlyT1 compound under the same anesthetic regimen (data not shown).

Fig. 4
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Dynamic ASL measurements on CBF in rat during administration of pharmacological agents known to perturb the NMDA system. Plots show temporal evolution of blood flow following administration of d-serine and a GlyT1 inhibitor, with and without blockade of the NMDA glycine site via a highly selective glycine site blocker. Scale bars represent 10 min and 10% change in CBF with respect to baseline (dotted line). Solid arrows denote injections

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Given the positive response in perfusion elicited by d-serine, it is surprising that inhibition of GlyT1 produced a decrease in perfusion. Nevertheless, the NMDA receptor component of this response was demonstrated by the prior administration of the highly selective glycine site blocker. In addition, a similar perfusion decrease occurred after administration of a structurally different GlyT1 inhibitor, supporting that this response is a result of GlyT1 inhibition and not off-target activity. While further work is necessary to more fully characterize the effects of d-serine and the GlyT1 inhibitor on CBF, these data suggest that GlyT1 inhibitors indeed affect the NMDA receptor system. Furthermore, these data show that changes in cerebral perfusion, measured in vivo with noninvasive ASL can provide a physiologic biomarker for assessment of the pharmacodynamic effects of novel psychoactive compounds.

The second example is an fMRI investigation of the mechanism of action of systemic lidocaine on noxious electrical stimulated CNS activity. Systemic lidocaine (e.g., intravenous infusion) has been increasingly used in the management of chronic pain syndromes such as neuropathic pain (Dirks et al. 2000; Bath et al. 1990; Mao & Chen 2000; Geha et al. 2007) since it was first introduced in 1961 for postoperative pain relief (Bartlett & Hutaserani 1961). The mechanism of action by which systemic lidocaine relieves neuropathic pain is unclear (Mao & Chen 2000). Neuropathic pain can result from peripheral nerve damage. The damaged nerve fibers spontaneously generate neural activity, ectopic discharges (Devor 1991), without activation of peripheral receptors. Previous studies have shown that systemic lidocaine suppresses ectopic discharges without blocking peripheral nerve conduction of the normal neural activity induced by electrical and mechanical stimulations (Puig & Sorkin 1995; Devor et al. 1992), suggesting that the action site of systemic lidocaine is the damaged nerves rather than the normal peripheral nerves which conduct the neural activity. We describe below, an fMRI approach to test if systemic lidocaine can block the peripheral nerve transmission of normal activity induced by electrical stimulation.

Lumbar spinal cord fMRI was performed in a 2 mm sagittal slice thickness covering the bilateral dorsal horns, before, during and after infusion of lidocaine at the rate of 1 mg/kg/min (volume rate: 0.1 ml/kg/min) over ∼20 min. As a control study, saline was infused at the same volume rate in separate rats. Figure 5 shows fMRI results from one animal with saline infusion and one animal with lidocaine infusion. Fig. 5a features the activation maps of saline and Fig. 5b of lidocaine infusion before, during, and after infusion. Under identical statistical criteria, the activation volumes and activation strength during the lidocaine infusion are smaller compared to those before infusion and those after stopping infusion, indicating that the systemic lidocaine does suppress the stimulation-induced activation in spinal cord. As expected, no significant difference in the activation maps is observed for the rat receiving saline infusion (Fig. 5a).

Fig. 5
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BV-weighted fMRI demonstrating effect of intravenous infused lidocaine on the noxious bilateral hind paw stimulation-induced fMRI signals in spinal cord. Illustrative data from a saggittal slice covering dorsal horns in two representative animals, receiving intravenous infusion of saline (a) and lidocaine (b), respectively. Negative ΔS/S changes (blue/violet as indicated by the color bar) detected in the BV-weighted fMRI indicate an increase in spinal blood volume. For saline infusion (a), robust activations can be detected, and are highly reproducible before, during, and after the saline infusion with regard to both the activation pattern and activation strength. For lidocaine infusion (b), the activation becomes weaker during the infusion of lidocaine, and slowly recovers after stopping infusion. Two horizontal lines indicate the positions of disk between T13 and T12 (top) and disk between L1 and L2 (bottom). R rostral, V ventral

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The suppression effect by lidocaine in the spinal cord fMRI signal measured can be attributed to either a peripheral nerve conduction blockage, and/or to an effect of lidocaine directly on the spinal cord. However, previous studies have excluded the spinal effect of lidocaine due to the fact that the intrathecal lidocaine injection could not suppress the tactile allodynia in neuropathic rats (Chaplan et al. 1995) and the lower limb postamputation stump pain in patients (Jacobson et al. 1990). Therefore the data in this study appear to suggest that the suppression of fMRI activity is likely due to the peripheral nerve conduction blockage. In conclusion, systemic lidocaine, which is believed to only block the peripheral nerve transmission of abnormal neural activity (ectopic discharge) originating from the damaged peripheral nerves, also appears to block the peripheral nerve transmission of normal neural activity induced by transcutaneous noxious electrical stimulation.

Concluding Remarks

In this chapter, we presented a practical approach to using fMRI for drug discovery using animal models. While fMRI can provide valuable information relating to CNS drug discovery, other MR methodologies such as volumetric MRI, perfusion and diffusion MRI, MR angiography, magnetic resonance spectroscopy (MRS), and the use of targeted contrast agents are being increasingly used by the pharmaceutical industry to help answer questions in nearly all disease areas. Rapid advances in new techniques and instrumentation in magnetic resonance, and increasing investments made by the pharma on imaging methodologies should result in a rapid growth of imaging based research in the drug discovery process.