From anatomy to the targetContributions of magnetic resonance imaging to preclinical pharmaceutical research.код для вставкиСкачать
THE ANATOMICAL RECORD (NEW ANAT.) 265:85–100, 2001 RESEARCH ARTICLE From Anatomy to the Target: Contributions of Magnetic Resonance Imaging to Preclinical Pharmaceutical Research NICOLAU BECKMANN,* THOMAS MUEGGLER, PETER ROLAND ALLEGRINI, DIDIER LAURENT, AND MARKUS RUDIN In recent years, in vivo magnetic resonance (MR) methods have become established tools in the drug discovery and development process. In this article, the role of MR imaging (MRI) in the preclinical evaluation of drugs in animal models of diseases is illustrated on the basis of selected examples. The individual sections are devoted to applications of anatomic, physiologic, and “molecular” imaging providing, respectively, structural-morphological, functional, and target-specific information. The impact of these developments upon clinical drug evaluation is also briefly addressed. The main advantages of MRI are versatility, allowing a comprehensive characterization of a disease state and of the corresponding drug intervention; high spatial resolution; and noninvasiveness, enabling repeated measurements. Successful applications in drug discovery exploit one or several of these aspects. Additionally, MRI is contributing to strengthen the link between preclinical and clinical drug research. Anat Rec (New Anat) 265:85–100, 2001. © 2001 Wiley-Liss, Inc. KEY WORDS: biomedical imaging; imaging; magnetic resonance imaging; MRI; functional MRI; fMRI; pharmacologic MRI; pharmaceutical research; drug development MRI IN THE DRUG DISCOVERY/ DEVELOPMENT PROCESS The rapid advance of imaging techniques such as magnetic resonance Dr. Allegrini earned his PhD in Biochemistry at the University of Bern, Switzerland. He was a postdoctoral fellow at the Biocenter of the University of Basel from 1981 to 1985. He now heads an MRI laboratory at Novartis Pharma. In 1995, during a sabbatical leave, he spent 6 months at the Max-Planck-Institute for Neurological Research in Cologne. Dr. Beckmann received his BSc in Physics and MSc in Applied Physics at the University of São Paulo, Brazil, and from there was an Alexander von Humboldt Fellow at Siemens Medical Systems in Erlangen, Germany. After receiving his PhD in Biophysics at the Biocenter of the University of Basel in 1990, he was appointed Director of Physics of the MR Center of the University of Basel. Since 1993, he has headed an MRI laboratory at Novartis Pharma. Dr. Laurent received a PhD in biology at the University of Grenoble, France. He conducted postdoctoral research in the area of in vivo MRI and NMR spectroscopy at Ciba-Geigy Pharmaceuticals in Basel, and eventually at Yale University. Since 1999, he has headed an MRI laboratory at Novartis Pharma. Mr. Müggler received his BSc in Biology from the University of Bern in 1999. © 2001 Wiley-Liss, Inc. imaging (MRI; see Box 1 for abbreviations) has made them indispensable tools in clinical diagnosis. Whereas the drive for method development was clearly given by clinical applications, recently similar approaches became available for experimental studies in research animals as well. The major issues that have to be addressed in animal imaging are the demands for high spatial resolution and the associ- He is currently doing PhD work in Biophysics at the Biocenter of the University of Basel. Dr. Rudin received his PhD in Physical Chemistry in 1981 at the Federal Polytechnic Institute (ETH) in Zurich. He then worked on a joint project of the ETH Zurich and the Swiss Institute for Water Pollution Control, and thereafter at the Biocenter of the University of Basel. He became head of an MRI laboratory at Novartis Pharma in 1983. He is currently head of the Analytical and Imaging Sciences Unit at Novartis Pharma and Associate Professor for Biophysics at the University of Basel. *Correspondence to: Dr. Nicolau Beckmann, Novartis Pharma Ltd., Core Technologies Area, Analytics and Imaging Sciences Unit, WSJ-386.209, CH-4002 Basel, Switzerland. Fax: ⴙ41-613243889; E-mail: nicolau.beckmann@ pharma.novartis.com ated issues of improved sensitivity. From a technologic point of view, the status of clinical and animal MRI today can be considered equivalent. It is precisely the link between preclinical and clinical applications that renders MRI so attractive in pharmacologic research. In this review, we illustrate the role of MRI in the preclinical evaluation of drugs in animal models of diseases. Animal models are used in several phases of the drug development process. The first steps are the identification and validation of a potential drug target. Genetically engineered animals are being increasingly used in this early phase. Later in the process, the safety and efficacy of drug candidates are evaluated in animal models of human diseases. Ideally, in order that study paradigms be easily transferable to clinical drug development, questions concerning drug absorption, distribution, efficacy, metabolism, and elimination should be addressed in a noninvasive manner with high sensitivity and spatial resolution. For most drugs, average tissue con- 86 THE ANATOMICAL RECORD (NEW ANAT.) RESEARCH ARTICLE Box 1. Abbreviations used in this article ACPL ⫽ antibody-conjugated paramagnetic liposomes, ADC ⫽ apparent diffusion coefficient, BAL ⫽ broncho-alveolar lavage, BOLD ⫽ blood-oxygenation-level-dependent, CA ⫽ contract agent, CBF ⫽ cerebral blood flow, CBV ⫽ cerebral blood volume, DWI ⫽ diffusion-weighted imaging, EAE ⫽ experimental autoimmune encephalitis, ECG ⫽ electrocardiogram, ETR ⫽ engineered transferrin receptor, fMRI ⫽ functional magnetic resonance imaging, ICAM-1 intercellular adhesion molecule-1, Gd ⫽ gadolinium, ITP ⫽ interphalangeal, i.v. ⫽ intravenous, mBSA ⫽ methylated bovine serum albumin, MCA ⫽ middle cerebral artery MION ⫽ centration is too low to be detected by using MR methods. Only nuclear medicine techniques, like gamma scintigraphy, single-photon emission computer tomography (SPECT) or positron emission tomography (PET) possess the required sensitivity for exposure and pharmacokinetic studies. However, these methods are limited by relatively poor resolution (ⱖ1 mm) and lack of chemical specificity, i.e., the inability to distinguish whether the emitting radionuclide is bound to the parent drug molecule or to a metabolite thereof. The principal assets of MRI are high spatial resolution, of the order of 100 m for rodent studies, and the excellent soft tissue contrasting capabilities. The MR signal is governed by several parameters, e.g., proton density, relaxation times (T1, T2, T2*), proton exchange rates, water diffusion, macroscopic motion (blood flow), which depend on the biophysical properties of the tissue (see Mori and Barker, 1999 for a general technical explanation of the principles of MRI, including relaxation times). This wealth of information renders MRI a valuable tool for diagnosis, tissue staging, and in vivo morphometry, for obtaining physiologic and functional readouts, and for deriving metabolic and target-specific tissue characteristics. In view of the increased life expectancy in industrialized countries the prevalence of neurodegenerative (Parkinson and Alzheimer diseases) and cardiovascular disorders, diabetes, cancer, or bone and joint diseases may also increase. The use of noninvasive imaging techniques to detect monocrystalline iron oxide nanoparticles, MPTP ⫽ 4-phenyl1,2,3,6-tetrahydropyridine hydrochloride, MR ⫽ magnetic resonance, MRA ⫽ magnetic resonance angiography, MRI ⫽ magnetic resonance imaging, MRS ⫽ magnetic resonance spectroscopy, OA ⫽ ovalbumin, PcommA ⫽ posterior communicating artery, PET ⫽ positron emission tomography, pMCAO ⫽ permanent MCA occlusion, RA ⫽ rheumatoid arthritis, SPECT ⫽ single-photon emission computer tomography, TE ⫽ echo time, TOF ⫽ time-offlight and monitor disease progression and to evaluate therapy response in animal models of these diseases will become increasingly important for the pharmaceutical industry. Because repetitive measurements are feasible, the inter-individual variance can be reduced by using each animal as its own control, thereby enhancing the statistical power of the experiments. The use of MR methods in pharmaceutical research has been already extensively reviewed (Rudin et al., 1995, 1999; Beckmann et al., 2000). The primary role of MR methods in pharmacologic research is to study drug effects on tissue morphology, physiology and (endogenous) biochemistry. The strengths of the approach are versatility, allowing a comprehensive tissue characterization, high spatial resolution, and noninvasiveness. The poor sensitivity of MR largely prevents its use for pharmacokinetic studies. Here we will illustrate the use of MRI methods in pharmacologic research on the basis of selected examples. The individual sections are devoted to applications of anatomic, physiologic, and “molecular” imaging, providing structural-morphologic, functional, and target-specific information. The impact of these developments upon clinical drug evaluation will also be briefly addressed. ANATOMIC IMAGING: TISSUE STAGING AND IN VIVO MORPHOMETRY MRI is inherently a three-dimensional (3D) technique providing cross-sectional images as well as full 3D data of body regions (Mori and Barker, 1999). Depending on the spatial resolution required and the imaging protocol applied, acquisition times for one image range from typically 80 ms to 1 hr. It is evident that longer acquisition times are necessary to improve the spatial resolution. Because animals need to be kept immobilized and anesthetized, total investigation times including preparation are usually set to 1 hr at maximum. Anatomic imaging constitutes the basis of pharmacologic applications of in vivo MR techniques. However, rather than being competitive to histology, MRI provides complementary information. Histology is and will remain the gold standard of anatomy in most pharmaceutical preclinical studies. Anatomic MRI becomes indispensable when noninvasiveness is an essential requirement, e.g., in longitudinal studies in chronic diseases, especially in those involving the use of transgenic animals and higher species. Moreover, due to the multiparametric signal dependence, MRI provides a comprehensive characterization of soft tissue pathologies. With this in mind, we will present some examples to illustrate the benefits of using anatomic MRI for the characterization of disease models and therapeutic interventions. Degenerative Joint Diseases Chronic diseases affecting the joints, such as rheumatoid arthritis (RA) and osteoarthritis, are a major medical challenge with the aging of the population, and an efficient therapy has yet to emerge. Due to its excellent softtissue contrast, allowing differentiation of bone, cartilage, tendons, synovium, muscle, and adipose tissue, RESEARCH ARTICLE THE ANATOMICAL RECORD (NEW ANAT.) 87 plified the location of the lesions, as well as the comparison with histology. The main advantage of applying MRI in the first example was the fact that one parameter, the joint gap volume, served as biomarker to monitor joint destruction and the effect of drug treatment. Additionally, assessments could be made in several joints of the hind paw. Quantitative assessment of individual joints by using histologic methods would have been very labor-intensive and time consuming. Alternatively, assessing paw swelling by measuring the external thickness of the hind paws as indicator of RA was of limited use, because swelling receded before significant structural changes were observed in the joints (Beckmann et al., 1995, 1998). In the second example, histologic assessment was carried out at the end of the Figure 1. Collagen-induced arthritis in the rat. High-resolution magnetic resonance image of interphalangeal joints of mid toes from an untreated and a Neoral- (cyclosporine-) treated rat. Images extracted from 3D data sets acquired 60 days after collagen administration (pixel size, 94⫻81⫻60 m3; acquisition time, 54.6 min). Adapted from Beckmann et al. (1998) with permission of the publisher. MRI has become the method of choice in the diagnosis of joint diseases in humans. (For information on MRand CT-microscopic imaging of bone in osteoporosis, see Borah et al. 2001.) MRI has also been used in animal models of RA (Waterton et al., 1993; Beckmann et al., 1995, 1998; Dawson et al., 1999). Animal studies, especially in small rodents, demand high spatial resolution and reproducibility of joint positioning in different imaging sessions. 3D imaging protocols allow reproducible monitoring of the complex joint architecture necessary for longitudinal studies. Proper coregistration of the images is feasible by postprocessing. In addition, 3D images are less affected by partial volume artifacts due to the smaller slice thickness achieved, improving the anatomic definition in the images. Figure 1 shows results obtained on a model of RA in which rats were immunized with type II bovine collagen (Beckmann et al., 1998). The images on the left side, extracted from 3D data sets acquired 60 days after collagen injection, depict the proximal interphalangeal (ITP) joints of the mid toes of hind paws of untreated and treated rats (Neoral 15 mg/kg per day p.o. for 42 days, starting 14 days after immunization). Volumes of the joint gaps, used as a marker to assess joint destruction by the arthritic process, are shown on the right side. Pathomorphologic changes associated with the collagen-induced arthritic process, e.g., increase of joint space and cartilage and bone erosion, have been observed in the control group. In contrast, the joint architecture remained unaltered in drug-treated rats. In the second model, RA was induced by intra-articular injection of methylated bovine serum albumin (mBSA) into the knee of rabbits that had been presensitized to the same antigen. High-resolution 3D MRI has been applied to investigate chronic joint damage in this model (Dawson et al., 1999). Inflammatory changes, such as soft-tissue swelling (edema formation), have been observed initially. Hard tissue (cartilage and bone) damage occurred at later time points (Figure 2). Use of 3D MRI was particularly important because the position of the lesions differed from animal to animal; proper selection of the orientation of the slices by using landmarks in the 3D data sets significantly sim- The primary role of MR methods in pharmacologic research is to study drug effects on tissue morphology, physiology, and (endogenous) biochemistry. study, with the position of the histologic sections being chosen based on the 3D MR images. This was especially important in preparing histologic sections of the damaged joints. The excellent correlation between histology and MRI indicated that the technique could be reliably applied to follow the disease process in this model of RA in rabbits, allowing to address questions concerning the effectiveness of therapy over extended treatment periods. Lung Inflammation From the MRI standpoint, the living lung is one of the most challenging organs to image. Because of the low water density of approximately 20 –30% (Harris and Heath, 1986), the proton density is significantly lower than in other tissues. Consequently, it is harder to ob- 88 THE ANATOMICAL RECORD (NEW ANAT.) RESEARCH ARTICLE Figure 2. Antigen-induced arthritis in the rabbit knee. Two sagittal sections extracted from high-resolution three-dimensional magnetic resonance image data sets (acquisition time, 31 min; pixel size, 219⫻219⫻250 m3) acquired from the right knee before (day 0) and at several time points relative to intra-articular injection of methylated bovine serum albumin. F, femur; T, tibia; M, meniscus; Se, sesamoid bone; Ef, synovial fluid effusion; Er, hard tissue erosion. Adapted from Dawson et al. (1999) with permission of the publisher. tain good imaging data. Moreover, due to the difference in magnetic susceptibilities between lung tissue and the air that comprises approximately 80% of pulmonary volume, local inhomogeneities in the magnetic field are produced, resulting in very short T2 and T2* relaxation times. In addition, cardiac and respiratory movements can cause severe image artifacts. These problems are more evident in rodents because of the higher cardiac and respiratory rates and the necessity to acquire images of improved resolution. To detect signal from lung parenchyma, especially at high fields, nonstandard techniques need to be used for considerably reducing the echo times (TEs) (Bergin et al., 1991). Furthermore, respiratory and cardiac motion has to be taken into account. For instance, by using projection encoding MRI protocols combined with scan-synchronous ventilation, highresolution images of the rat (Gewalt et al., 1993) and the guinea pig lung (Shattuck et al., 1997) have been obtained. An easier approach based on the gradient-echo sequence was used recently for the detection of lung inflammation after allergen challenge in the rat (Beckmann et al., 2001a). Potential image artifacts caused by cardiac and respiratory movements were suppressed by averaging. Neither respiratory nor ECG triggering was applied, and the rats could respire freely during data collection. At a TE of 2.7 ms, the signal from lung parenchyma was too weak to be detected at 4.7 T. The absence of detectable lung parenchyma signal in combination with a background free of artifacts provided high contrast-to-noise ratio for the detection of edema associated with pulmonary inflammation. This is clearly evident in Figure 3A, which reveals edema formation detectable 6 hr after intratracheal challenge with 0.3 mg/kg ovalbumin (OA). The edema progressed to a maximum at 48 hr after OA exposure and was clearly receding by 72 hr. The mean time course of edema formation after challenge with saline or different doses of OA is shown in Figure 3B. A dose-related response was observed, since higher doses of OA produced larger edema and, after 1 mg/kg OA, the maximum edema occurred at 24 hr, whereas for lower doses the maximum appeared later, around 48 hr after challenge. With the doses used, edema was observed for approximately 100 hr. No edema was detected after application of saline. MRI also revealed that budesonide, a potent glucocorticosteroid, could significantly accelerate edema resolution if given 24 hr after allergen challenge (Figure 3C). An important finding in this study was that changes in edema volume measured by MRI and protein concentration (a marker for plasma extravasation into the lungs) in the lung RESEARCH ARTICLE THE ANATOMICAL RECORD (NEW ANAT.) 89 fluid obtained by bronchoalveolar lavage followed a similar time course (Beckmann et al., 2001a). Thus, after the pulmonary edema formation, noninvasive MRI provides a reliable means for assessing pulmonary inflammation after an allergen challenge. Furthermore, therapy response can be evaluated in individual animals, illustrating the potential of MRI for the profiling of anti-inflammatory drugs both in animal models of asthma and in the clinic. Phenotyping Transgenic Animals The study of the effects of specific gene products on the pathophysiology has been enabled by the availability of knockout/transgenic technology in the mouse, allowing manipulation of the expression of specific genes and their particular translational products in this species. In the context of modern pharmacologic research and drug development, transgenic mice are of importance both as disease models and for target validation. Phenotyping of genetically engineered animals is essential, preferentially by using a noninvasive method. MR techniques are being increasingly used to assess phenotypes in models of neurodegenerative disorders (Cohen et al., 1999; Ferrante et al., 2000), cardiovascular disorders (Franco et al., 1999; Chacko et al., 2000; Omerovic et al., 2000; Wiesmann et al., 2000), metabolic disorders (Clapham et al., 2000), and stress (Beckmann et al., 2001b). Currently, several centers are being established worldwide with the aim of phenotyping transgenic mice by using high throughput MR techniques. The following example illustrates the advantages of using MRI in this area. Numerous genes are induced by ce- Figure 3. Lung inflammation in the rat due to allergen challenge. a: Representative sections through the rat chest, obtained previous to and at various time points after i.t. instillation of 0.3 mg/kg ovalbumin (OA). Mean edema volume (⫾ SEM) as a function of time after i.t. challenge with (b) saline or various doses of OA, (c) 0.3 mg/kg OA in untreated animals and animals treated i.t. with budesonide, administered 24 hr after challenge. Adapted from Beckmann et al. (2001a) with permission of the publisher. 90 THE ANATOMICAL RECORD (NEW ANAT.) Figure 4. Coronal maximum intensity projections of three-dimensional angiograms (16.4min acquisition time) of the brains of mice from three different strains. The middle cerebral arteries (MCAs) are indicated by red arrows, whereas the anastomoses at the level of the posterior communicating artery (PcommA) are indicated by yellow arrows. Although the CD1 mice showed well-developed, albeit unilateral, PcommA, this anastomosis was less well developed in C57BL/6 and SV-129 mice. The MCAs in the SV-129 strain were significantly shorter than in other strains. Adapted from Beckmann (2000) with permission of the publisher. rebral ischemia, such as genes encoding the heat shock protein hsp72 (Kinouchi et al., 1993), the cytokines interleukin-1, interleukin-6, and tumor necrosis factor-␣ (Buttini et al., 1996; Wiessner et al., 1993), and the immediate-early genes c-fos, c-jun, and junB (Kinouchi et al., 1994). Transgenic mice are being used to investigate ischemia-induced genomic responses in the brain, to clarify the mechanisms of inherent degenerative or protective processes, and to define targets for specific modulations of these cellular responses. Several approaches exist to induce focal cerebral ischemia in mice, e.g., permanent occlusion of the middle cerebral artery (MCA) and transient occlusion of the MCA with an intraluminal thread. The last is of special interest because of the possibility of studying the consequences of reperfusion. In this method, a thread is advanced from the internal carotid artery into the anterior cerebral artery, until its tip covers the MCA. Because many factors can lead to inconsistent vascular occlusion, e.g. variable thread diameter and vessel size, as well as biological variability, reproducible ischemia requires precise standardization of experimental conditions. Differences in vascular anatomy have to be taken into account when cerebral ischemia is produced in a transgenic or knockout mouse (Kitagawa et al., 1998; Maeda et al., 1998). In particular, it has been shown that the patency of the posterior communicating artery (PcommA), which connects the superior cerebellar and the posterior cerebral arteries, is a crucial determinant of ischemia in the PcommA territory after intraluminal suture occlusion (Kitagawa et al., 1998). This patency, although being always present in rats, is highly variable and strain-dependent in mice (Barone et al., 1993). Furthermore, the supplying territory of the MCA, which determines the size of the infarcted area after intraluminal occlusion, has also been shown to be straindependent (Maeda et al., 1998). Recently, high-resolution MR angiography (MRA) has been used to verify the success of transient MCA occlusion and reperfusion in rats (Reese et al., 1999) and mice (Beckmann et al., RESEARCH ARTICLE 1999). By using 3D time-of-flight (TOF) techniques (based on the suppression of stationary tissue by saturation of the longitudinal magnetization; the degree of signal enhancement strongly depends upon the velocity of the blood and the interval between radio-frequency pulses), the cerebral arterial vascular structure could be detected with high contrast in both species in acquisition times ranging between 8 and 25 min, without the application of contrast agents. Also, MRA revealed highly variable arterial cerebrovascular structures in mice from different strains and within the same strain (Beckmann, 2000). Of special interest was the detection of differences at the level of the PcommA. Furthermore, SV-129 mice showed significantly shorter MCAs compared with other strains (Figure 4). C57BL/6 and SV-129 mice have been the most widely used parent strains for the production of mutants. Because of the different vascular architectures of these strains, differences in infarct volume after targeted gene mutation could well be confused with differences in the genetic background of the parent strains. Therefore, it is important to confirm that independent variables, as differences in vascular architecture, do not interfere with the result of gene targeting. Visualization of the vascular architecture by MRA provides a means of detecting and refuting the existence of such interactions. Also, noninvasively assessing whether and on which side of the circle of Willis the PcommAs are present could be of help in selecting the animals and/or the side for performing the MCA occlusion. Presurgery MRA evaluation could potentially help to improve the reproducibility of murine cerebral infarction studies. In summary, anatomic MRI has become an indispensable tool for qualitative and quantitative tissue characterization, including the identification of pathomorphologic changes. The method is inherently 3D and yields highly accurate morphometric data. PHYSIOLOGIC MRI: DYNAMIC ASSESSMENT OF FUNCTIONAL PARAMETERS Physiologic MRI refers in a broader sense to the mapping of physiologic RESEARCH ARTICLE THE ANATOMICAL RECORD (NEW ANAT.) 91 nal-to-noise ratio of the signal of the gas in the lungs, also that the background signal is nonexistent. Pharmacologic Stimulation of the Central Nervous System Figure 5. Hyperpolarized 3He coronal slice of the rat lungs (right) with an enlarged view of the right upper lobe from the same image (left). This radial projection was acquired during multiple breath holds after full inspiration. Courtesy of G.A. Johnson, Duke University. parameters such as lung ventilation, tissue perfusion and oxygenation, myocardial motion, tracer clearance and uptake, metabolism, and neural activity. Its relevance for the characterization of pathologies lies in the fact that changes in physiologic parameters generally precede morphologic alterations. Performing functional studies in small rodents is particularly challenging due to the demands on spatial and temporal resolution as physiologic processes (e.g., heart rate, ventilation, bolus passage in perfusion studies) are considerably faster than in humans. The following examples should illustrate that, nonetheless, functional studies with MRI are playing an increasing role in pharmacologic research. Lung Ventilation Gas exchange is the major function of the lung. Matched distribution of the regional pulmonary blood flow (perfusion) and ventilation is necessary for this process to occur efficiently. The large amount of ventilation in the lungs is matched by a correspondingly high perfusion. The balance between pulmonary perfusion and ventilation can be altered in disease states. Accurate assessment of perfusion and ventilation is especially important in the evaluation of patients with suspected pulmonary embolism, a common and life-threatening disorder that can be treated by anticoagulants. Recently, MRI of the lungs after in- halation of hyperpolarized gases (Albert et al., 1994; Middleton et al., 1995; Black et al., 1996; Kauczor et al., 1996; Wagshul et al., 1996; de Lange et al., 1999) has aroused interest among the medical community because ventilation defects can be clearly identified on the images. In addition, dynamic ventilation images, which provide regional information about the gas dynamics in the lungs, can be obtained with high temporal resolution with this technique (Johnson et al., 1997; Saam et al., 1999; Viallon et al., 1999, 2000). Information obtained from dynamic ventilation images are complementary to static, breath-hold ventilation images. These methods are of interest in the context of pharmacologic research also, in models of pulmonary diseases like asthma and emphysema in small rodents (Hedlund et al., 1999). To perform such studies, mechanical ventilation becomes mandatory to maintain normal gas exchange and to control breathing motion. Furthermore, ventilators must be able to deliver hyperpolarized gases in synchrony with the imaging process without contributing to unnecessary depolarization. Figure 5 shows a hyperpolarized 3He coronal slice of the lungs of a rat, obtained by triggering the image acquisition by using an MRcompatible ventilator (Hedlund et al., 2000). The main advantage of using hyperpolarized gases is the good sig- In normal mammalian brain, activity is closely coupled to metabolic rates of oxygen and glucose and to cerebral blood flow (CBF). These couplings are the basis for methods used to noninvasively assess regional brain activity, such as PET and functional MRI (fMRI). The major focus of fMRI is presently directed toward human brain mapping (Zeineh et al., 2001). However, the high spatial and temporal resolutions achievable make fMRI an attractive tool to study brain function in laboratory animals as well. Local activation in sensory cortical areas has been demonstrated in healthy, anesthetized rats after electrical, somatosensory stimulation during normocapnia (Gyngell et al., 1996; Kerskens et al., 1996; Yang et al., 1996) and hypercapnia (Bock et al., 1998), and to monitor functional recovery after cardiac arrest (Schmitz et al., 1998). In another attractive application of fMRI in the rat brain, hemodynamic changes reflecting local and temporal changes of brain activities after administration of CNS stimulatory or inhibitory drugs can be measured. Such studies may contribute to a better understanding of the sites and mechanisms involved in the action of CNS drugs. Brain stimulations by physical or pharmacologic means, once elaborated and characterized, could be used for both differential diagnosis of neurologic or psychiatric disorders, as well as for evaluating the efficacy of therapeutic interventions. The feasibility of this approach has been demonstrated by several groups. Stein et al. (1998) used fMRI to demonstrate the acute CNS effects of nicotine in smokers. Nicotine induced a dose-dependent increase in neuronal activity in a distributed system of brain regions, including the nucleus accumbens, amygdala, cingulate, and frontal lobes. In a rat model of Parkinson disease, in which the striatum was lesioned unilaterally by injection of 6-OH-dopamine, no response to am- 92 THE ANATOMICAL RECORD (NEW ANAT.) Figure 6. Regional activation in the rat brain by bicuculline. Changes in cerebral blood volume as a function of time for cortex, caudate putamen, thalamus, and cerebellum. Bicuculline infusion at a dose of 1 mg/kg per min for the first 2 min (gray bar) and 0.1 mg/kg per min for the subsequent 10 min (light gray bar). Adapted from Reese et al. (2000a) with permission of the publisher. phetamine injected systemically was observed in the lesioned area in contrast to the unlesioned striatum, an observation that was paralleled by PET data (Chen et al., 1997). A related study was carried out in rhesus monkeys with unilateral striatal 4-phenyl1,2,3,6-tetrahydropyridine hydrochloride (MPTP) lesions showing altered responses to stimulation with levodopa in the affected area (Chen et al., 1996). Recently, Reese et al. (2000a) described transient changes in local cerebral blood volume (CBV) in the rat brain caused by i.v. administration of the GABAA antagonist bicuculline. Upon infusion of bicuculline, regionspecific increases in CBV occurred, reflecting brain activity (Figure 6). During the first 2 min, increased CBV was predominant in the cortex, followed by increases in other brain areas. Ten minutes after the start of the infusion, a dominant response was observed in the thalamus, whereas CBV in the caudate putamen showed a biphasic response pattern (Reese et al., 2000a). The magnitude of the signal responses in all brain regions was dependent on the bicuculline dose. Similar results were obtained for the mouse brain (Mueggler et al., 2001). These rodent studies show that pharmacologic fMRI, displaying brain function at the highly specific level of drug-receptor interaction, should foster our understanding of normal and pathologic brain function (see also “Mapping of Ligand-Receptor Interactions,” below). However, because anesthesia can affect the results, e.g., when probing cortical responses in the brain due to somatosensory stimuli, the complex pharmacology of anesthetics as a result of their interaction with several different receptor systems (Franks and Lieb, 1999) has to be considered when testing compounds in fMRI experiments. Functional Studies in Stroke Research Cerebral infarction is one of the leading causes of mortality and morbidity in industrialized countries. Despite major research efforts in both academia and industry, there is to date no generally accepted therapy. Several animal models of infarct do exist. The application of various MRI approaches using the multiparametric character of MRI contrast mechanisms allows a comprehensive characterization of the pathophysiologic cascade (Figure 7). Determination of the link between the observed changes in MRI contrast parameters and physiologic events is essential to address mechanistic aspects. As an example, consider the permanent MCA occlusion (pMCAO) model in rats (Tamura et al., 1981), which is routinely used to test drugs with various mechanisms aimed at reducing the extent of cellu- RESEARCH ARTICLE lar damage in the acute phase of stroke (Sauter and Rudin, 1986). MRA allows to assess the success of vascular occlusion (Beckmann et al., 1999; Reese et al., 1999). Perfusion-weighted MRI provides information on changes in CBF (Rudin and Sauter, 1991; Kohno et al., 1995; Rudin et al., 1997), whereas methods exploiting blood-oxygenation-level– dependent (BOLD) contrast assess relative tissue oxygenation (Roussel et al., 1995). Diffusionweighted imaging (DWI) enables early (within minutes after MCA occlusion) detection of cerebral changes preceding infarction (Moseley et al., 1991; Mori and Barker, 1999). A significant decrease of the apparent diffusion coefficient (ADC) of water is observed in the ischemic region. The observed changes in ADC have been associated with the formation of cytotoxic edema: as the intracellular ADC value is smaller than the extracellular, an increase of the intracellular volume fraction leads to a reduction of the total ADC (Benveniste et al., 1992). With T2-weighted sequences, a high contrast between infarcted (edematous) and healthy tissue is obtained at 24 or 48 hr after MCA occlusion, allowing quantitative analysis of infarct volumes. fMRI studies involving, e.g., electrical or pharmacologic stimulation are used to probe the functional recovery of infarcted tissue after treatment. The time course of these parameters in a model of MCA occlusion in the rat has been discussed in detail by Hoehn-Berlage et al. (1995) and Rudin et al. (2001). In the pMCAO model of stroke, the effectiveness of a therapy is commonly judged from infarct size measurements, assuming that a reduction in infarct size results in reduction of the functional deficits. Reese et al. (2000b) evaluated the concept that structural integrity translates into functional integrity during the acute post-stroke period (24 hr). The functional integrity of the somatosensory cortex was assessed by detecting changes in local CBV after electrical stimulation of the forepaws and pharmacologic stimulation by bicuculline (see previous section). In sham-operated rats, simultaneous electrical stimulation of both forepaws led to similar activation of both somatosensory cortices, whereas in pMCAO animals, only the contralateral cortex RESEARCH ARTICLE THE ANATOMICAL RECORD (NEW ANAT.) 93 Figure 7. Middle cerebral artery (MCA) occlusion model of stroke in the rat. The multiparametric nature of MRI is explored to follow the course of pathophysiology in this model. Brain activity mapping was derived from experiments involving electrical stimulation of both forepaws. FMRI, functional MRI. 94 THE ANATOMICAL RECORD (NEW ANAT.) RESEARCH ARTICLE showed an fMRI response. Similarly, in pMCAO rats treated with the calcium antagonist isradipine, functional activation after bilateral electrical stimulation was only detected in the contralateral somatosensory cortex, despite the normal appearance of the ipsilateral cortex in T2-weighted and diffusion-weighted images. Furthermore, fMRI responses to pharmacologic stimulation with bicuculline were virtually absent in the ipsilateral somatosensory cortices both in vehicle- and isradipine-treated animals (Reese et al., 2000b). These results correlated well with behavioral studies (beam-walking test) carried out with the same rats. Thus, structural integrity in the somatosensory cortex revealed by anatomic MRI does not necessarily translate into preservation of function in the acute (24 hr) postocclusion period. Probing functional integrity by fMRI in addition to structural assessments by MRI could become an integral part of routine drug testing in stroke models. Kidney Transplantation Organ transplantation is currently the preferred procedure in end-stage organ disease; although, because the introduction of cyclosporine immunosuppressive therapy acute rejection can be well managed, chronic rejection remains the main complication. This rejection involves both the reaction of the host to the graft (mainly T-cell mediated and also antibody — B-lymphocyte-driven) and reactions in the graft toward injury (vasculopathy or graft vessel disease in, e.g., kidney and heart grafts, bronchiolitis obliterans in lung grafts). The first reaction asks for improved immunosuppressants including tolerance-inducing approaches, the second one for interventions to protect the graft (e.g. endothelial cells) from damage like ischemia/reperfusion injury or for treatment of vessel disease (Olsen, 1991; Cook et al., 1998). An important animal model of kidney transplantation is orthotopic kidney allografting in rats by using the strain combination of DA into Lewis. This strain combination is generally considered to be very stringent, because of the major histocompatibility mismatch between donor and recipient, and the strong alloreactive potential of Lewis Figure 8. Noninvasive assessment of graft function in a DA to Lewis model of kidney transplantation (tx) by MRI. a: Perfusion MRI. A series of MR images is acquired sequentially with a time resolution of 800 ms per image. After acquiring baseline images, a contrast agent containing iron nanoparticles is injected i.v. during 1 sec. The signal attenuation is proportional to the perfusion in the organ. b: Clearance of an extracellular contrast agent (Gd-DOTA). Similarly to perfusion MRI, images are acquired sequentially (time resolution of 6 sec per image). After acquisition of baseline images, the contrast agent is injected i.v. during 2 sec. Signal enhancement for native, normal kidneys (left) and for a rejected kidney (right). Adapted from Beckmann et al. (2000) with permission of the publisher. rats. If left untreated, animals with acute rejection die at around day 11 due to the loss of graft function. Long-term survival of the transplant (up to 130 days) is achieved in recipients treated with cyclosporine starting from the day of transplantation until day 14 (Borel, 1989). With the aim of determining the efficacy of immunosuppressive treatments, anatomic and perfusion MRI as well as MR renography were used as a means to assess noninvasively graft status in this model (Beckmann et al., 1996, 2000; Schuurman et al., 1996; Baumann and Rudin, 2000). Perfusion MRI involving the i.v. ad- RESEARCH ARTICLE THE ANATOMICAL RECORD (NEW ANAT.) 95 procedures to analyze spectra of body fluids, e.g., of urine, to evaluate the nephrotoxic effects of drugs (Anthony et al., 1994; Holmes et al., 1995). MOLECULAR MRI: IMAGING THE TARGET Figure 8. (Continued) ministration of an intravascular contrast agent (CA) reveals that tracer concentration in a rejected kidney is significantly lower than that in native, normal kidneys (Figure 8A), demonstrating a compromised perfusion of the graft due, e.g., to vasculopathy. Furthermore, applying MR renography techniques (Baumann and Rudin, 2000), signal enhancements in the cortex and medulla of a rejected kidney after i.v. application of an extracellular CA were significantly smaller than those observed in native organs for the same amount of CA administered. In particular, almost no signal enhancement occurred in the pelvis, demonstrating that the clearance of the extracellular contrast agent was severely compromised in the rejected graft (Beckmann et al., 2000). Of importance is that changes in perfusion and tracer clearance occurred before anatomic changes. These functional parameters are, therefore, sensitive markers of early kidney rejection. Physiologic MRI allows the assessment of pathophysiological alterations. As these changes frequently precede structural pathologies, physiologic MR readouts have been shown to serve as early biomarkers of the disease process and of therapy response. Although not yet systematically pursued, anatomic and functional MRI, as well as MR spectroscopy readouts, may also add value to identifying toxicologic effects of drugs. An interesting approach in this regard is high-resolution MR spectroscopy in combination with pattern recognition MRI has evolved over the past decade from a radiologic technique to a method that provides information on physiologic tissue parameters, e.g., tissue blood flow or oxygenation. Presently, MRI is evolving toward molecular or target-specific imaging, i.e., the mapping of ligand-target interactions. Target-specific imaging providing direct insight into biological processes at the molecular or cellular level is an important aid in addressing fundamental questions in biomedical research. Because of its high sensitivity and wider availability, PET is currently (and for the foreseeable future) the method of choice in the area of molecular imaging. However, PET has the drawback of requiring the design and synthesis of a suitable ligand, which has to be prepared in situ because of the short halflife of the positron-emitting nuclei. Thus, in certain instances, MRI with its superior spatial resolution might be exploited either by following the PET concept of using magnetically labeled ligands or by mapping the consequences of a ligand-receptor interaction on physiological tissue parameters. Both avenues are going to be illustrated here. A strength of MRI is its ability to allow direct comparison of anatomic and molecular images. Magnetically Labeled Ligands Paramagnetic gadolinium (Gd) complexes such as Gd-DTPA, which penetrate the extracellular space and primarily alter the T1 values of tissues, and superparamagnetic iron oxide preparations, which exhibit a susceptibility that is two to three orders of magnitude larger than that of paramagnetic complexes, are the contrast agents commonly used in MRI. By coupling them to target-specific ligands, receptor-specific CAs have been synthesized. For instance, a liver-specific CA was obtained by coupling iron nanoparticles to asialofetuin, a ligand for the hepatic asialoglycoprotein receptor (Reimer et al., 1991). Coupling to secretin yielded pref- 96 THE ANATOMICAL RECORD (NEW ANAT.) Figure 9. In vivo magnetic resonance image of a mouse with engineered transferrin receptorpositive (ETR⫹, left) and ETR⫺ (right) flank tumors. Composite image of a T1-weighted spin-echo image with superimposed contrast changes after Tf-MION administration. Inserts: Histological analysis of ETR expression, showing iron staining in ETR⫹ (left) and ETR⫺ (right) tumor cells. Courtesy of R. Weissleder, Harvard University. Adapted from Weissleder et al. (2000) with permission of the publisher. erential accumulation of the CA in the pancreas because of the high affinity to the secretin receptors, which are abundant in exocrine pancreatic cells (Shen et al., 1996). Lewin et al. (2000) labeled stem and progenitor cells ex vivo with superparamagnetic iron oxide nanopar- ticles that had been coated with dextran covalently bound to peptide sequences from the HIV-derived Tat protein. The cells were injected into immunodeficient NOD/SCID mice. The Tat-derived sequences enabled the paramagnetic label to be efficiently internalized into target cells, RESEARCH ARTICLE which were tracked by MRI in harvested bone marrow and liver. Similarly, Bulte et al. (1999) tracked ex vivo cell migration and myelination in the spinal cord of myelin-deficient rats, into which magnetically labeled oligodendrocyte progenitor cells were transplanted. Cell migration could be easily detected by using 3D MRI, with a close correlation between the areas of contrast enhancement and the achieved extent of myelination. By allowing the direct imaging of cell trafficking, this approach may not only provide information for the optimization of stem cell-based therapies, but also advance our basic understanding of developmental biology, pathology, and general pharmacology. Real-time imaging of gene expression in vivo at high spatial resolution has been a long-lasting goal in molecular research. If such techniques were available, both endogenous and exogenous (e.g., gene therapy) gene expression could be studied in live animals and potentially in the clinic. The feasibility of such an approach was demonstrated recently by Weissleder et al. (2000), who used an engineered transferrin receptor (ETR) as a reporter that shuttled targeted superparamagnetic nanoparticles into cells. The internalizing receptor used was a human transferrin receptor that lacked the iron-regulating region and mRNA destabilization mo- Figure 10. Comparison of functional magnetic resonance imaging-derived maps of change in cerebral blood volume with the density of GABAA binding sites as derived from autoradiography (values taken from Chu et al., 1990). Adapted from Reese et al. (2000a). Reproduced with permission of the publisher. RESEARCH ARTICLE tifs in the 3⬘ untranslated region and, therefore, constitutively overexpressed high levels of the receptor in the cell (Casey et al., 1988). The receptor-targeted MR reporter consisted of 3-nm monocrystalline iron oxide nanoparticles (MION), sterically protected by a layer of low-molecular-weight dextran (Shen et al., 1993) to which human holo-transferrin was covalently conjugated (Tf-MION). To demonstrate that ETR expression correlated with increases in internalized Tf-MION, nude mice were implanted with both stably transfected (ETR⫹) and control transfected (ETR⫺) 9L gliosarcoma cells (Weissleder et al., 2000). Only transfected tumors (ETR⫹) overexpressed the engineered transferrin receptor and had more iron. This accumulation of iron was detected in vivo by T2- and T2*-weighted sequences, reflecting the increased transverse relaxation rate after cellular internalization (Figure 9). A further example is the visualization by MRI of leukocyte adhesion molecules in the intact brains of mice with experimental autoimmune encephalitis (EAE). Antibody-conjugated paramagnetic liposomes (ACPLs) targeted to intercellular adhesion molecule-1 (ICAM1), an endothelial leukocyte receptor up-regulated on cerebral microvasculature during EAE expression, were administered to diseased mice (Sipkins et al., 2000). High-resolution MRI of mouse brains ex vivo demonstrated that ACPL binding conferred significant enhancement compared with control images, suggesting that ACPLs can be used as CAs to visualize specific molecules expressed on vascular endothelium during disease. Another approach is the use of switchable CAs (Moats et al., 1997), which consist of a chelator with highaffinity binding to Gd and a galactopyranose residue positioned to block the remaining coordination site on the Gd ion from water. In this water-inaccessible conformation, the CA is inactive and does not strongly modulate T1. The enzyme ␤-galactosidase cleaves the sugar residue from the chelate, causing the irreversible transition of the CA to an active state, in which it increases relaxivity. By using switchable CAs, ␤-galactosidase mRNA expression in living X. laevis embryos was detected by MRI (Louie et al., 2000). By using genetic engineering approaches, ␤-gal can be THE ANATOMICAL RECORD (NEW ANAT.) 97 coupled to any promoter sequence; thus, in principle, the method described by Louie et al. (2000) could be a generic tool for imaging of gene expression. The problems that remain to be solved are cellular uptake of the CA and potential cytotoxicity. Mapping of Ligand-Receptor Interactions As mentioned previously, another avenue of molecular imaging consists in mapping the consequences of a ligand-receptor interaction on physiological parameters. The example already discussed above, namely the visualization of brain activation by bicuculline, is used as an illustration of this approach. Figure 10 presents a map representing the integrated CBV changes in the rat brain in the 20 min In pharmaceutical research, only MR applications that offer substantial advantages over existing technologies will be successful in the long term. after i.v. administration of bicuculline (Reese et al., 2000a). The density of GABAA binding sites taken from Chu et al. (1990) is also displayed for comparison. There was a good correlation between the degree of CBV changes and the receptor densities in the midand forebrain. Both values were high in frontal and parietal cortex as well as in thalamic nuclei, medium in the caudate putamen and lateral septal nucleus, and low in the hippocampal regions and other midbrain nuclei. Some discrepancy was observed in the cerebellum, where intermediate density of binding sites was reported for the molecular layer and very high density for the granule cell layer. By using this approach, the complex pharmacology of the ionotropic GABA receptor system (Bormann, 2000) would be amenable to an in vivo analysis. It is clear that molecular imaging will have a significant impact on biomedical research as well as on drug discovery and development (Rudin, 2000). Imaging the target; its location in cells, tissues, or in the body; its interaction with other molecules (e.g., substrate-target interactions, receptor occupancy); and the functional consequences thereof will be important not only in preclinical research but also in clinical work. The key success factor in such studies is the design of the appropriate labeled ligand rather than the imaging technology itself. The concerted interdisciplinary efforts of chemists, molecular biologists, and imaging experts are essential for making full use of the advantages offered by molecular imaging techniques. For MRI in particular, the main issue is coupling nanoparticles to bioactive peptidic ligands without significantly altering their receptor affinity and specificity. This method should be feasible in favorable cases as shown by the previous examples; the study of general drug-receptor interactions, however, is a major challenge. The principal limitations of molecular MRI are the inherent low sensitivity and the requirement of relatively bulky para- or superparamagnetic reporter groups. Despite that exciting MRI applications have been demonstrated to date, target-specific imaging will be the domain of more sensitive imaging modalities (e.g., PET and optical imaging). OUTLOOK MR methods have become an established tool in biomedical and pharmaceutical research, covering a broad range of applications from noninvasive morphologic measurements to comprehensive studies of tissue physiology and metabolism. Both in the clinical environment and in animal experimentation, MR methods are in competition with alternative, highly developed analysis tools. In pharmaceutical research, only MR applications that offer substantial advantages over existing technologies will be successful in the long term. The strengths of MR methodologies are noninvasiveness and the multiparametric character of the approach. This has been illustrated, for instance, with the stroke study (Figure 7), i.e., 98 THE ANATOMICAL RECORD (NEW ANAT.) information on local brain perfusion, local CBV, oxygenation deficit, cytotoxic and vasogenic edema, and functional responsiveness can be obtained with high spatial and temporal resolution in the same animal. Many other applications that exploit this versatility will be forthcoming. A prerequisite for studying chronic diseases both in humans and animals is noninvasiveness. In preclinical research, apart from the obvious statistical advantages when monitoring disease progression and therapy response in an individual, animal welfare and economical aspects need also to be taken into account. In long-term studies of chronic disorders — for which animals are expensive — it is economically attractive to use a noninvasive readout. The number of animals per study is reduced and the information obtained is refined, because additional studies can be performed in the same animal. This is especially important when working with higher species, e.g., nonhuman primates. Noninvasive technologies are also highly desirable when dealing with transgenic animals. Given the relevance of transgenics to modern biomedical and pharmaceutical research, the number of studies involving MR methods in the characterization of novel phenotypes in these animals will rapidly increase. The development of biomedical MR methods has been mainly driven by their clinical applications. In this context, preclinical research is lagging behind, mainly because the need for using noninvasive techniques is less imperative. However, a major advantage of using noninvasive technologies in animal research is the possibility of directly linking preclinical to clinical findings through relevant imaging biomarkers. This link will become even more important, as there is a strong need to further validate animal models of human disorders, e.g., multiple sclerosis, stroke, and dementias. The link between preclinical characterization of development compounds in animal models and clinical testing of drugs is also becoming tighter. In particular, there is a generalized strong tendency toward early pharmacodynamic studies in man, to assess whether new compounds are exerting the same pharmacologic ef- fects in patients as in animals. Such proof-of-concept studies aim at guiding the dose selection for the extensive and expensive clinical efficacy trials. Importantly, the safety of human subjects would not likely be compromised by these imaging methodologies. In this sense, it is foreseeable that preclinical studies involving MR methods and MR-derived biomarkers will play an increasing role in the validation and optimization of clinical protocols. MR methods will strengthen the link between the two intimately connected — however, in practice, often too distant — areas of preclinical and clinical research. 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