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TOOLS FOR ASSESSING SPINAL CORD INJURY AND REPAIR

Because the spinal cord is encased in the protective armor of the vertebrae, investigation of the site of the injury or the effects of potential therapies has required the development of a diverse set of research tools. In the past 40 years the rapid progress in the technologies available to perform experiments has largely been responsible for the great strides that have been made in understanding the basic principles of neuroscience. Studies with animal models have been instrumental in the rapid development of neuroscience and understanding of the biology of the spinal cord. The advent of cell culture techniques has provided a means to isolate and grow cells. Researchers can now isolate specific molecules and proteins and examine their roles in neuronal injury and repair in laboratory animals that mimic human spinal cord injuries. Recent advances in imaging techniques and methods for investigation of the actions of genes have advanced the understanding of spinal cord injuries even further. They also provide researchers with the tools that they need to examine changes in the spinal cord at the molecular and structural levels, for example, improving knowledge of the inhibitory conditions that serve as barriers to neuronal regeneration.

This chapter describes the important genetic and in vitro tools that have been developed to advance spinal cord injury research; the key animal models that are used to mimic human spinal cord injuries and the major limitations of the existing animal models; and the outcome measures that have been developed to assess spinal cord injuries and the effectiveness of experimental therapies, including the development of imaging technologies.



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Spinal Cord Injury: Progress, Promise, and Priorities 3 TOOLS FOR ASSESSING SPINAL CORD INJURY AND REPAIR Because the spinal cord is encased in the protective armor of the vertebrae, investigation of the site of the injury or the effects of potential therapies has required the development of a diverse set of research tools. In the past 40 years the rapid progress in the technologies available to perform experiments has largely been responsible for the great strides that have been made in understanding the basic principles of neuroscience. Studies with animal models have been instrumental in the rapid development of neuroscience and understanding of the biology of the spinal cord. The advent of cell culture techniques has provided a means to isolate and grow cells. Researchers can now isolate specific molecules and proteins and examine their roles in neuronal injury and repair in laboratory animals that mimic human spinal cord injuries. Recent advances in imaging techniques and methods for investigation of the actions of genes have advanced the understanding of spinal cord injuries even further. They also provide researchers with the tools that they need to examine changes in the spinal cord at the molecular and structural levels, for example, improving knowledge of the inhibitory conditions that serve as barriers to neuronal regeneration. This chapter describes the important genetic and in vitro tools that have been developed to advance spinal cord injury research; the key animal models that are used to mimic human spinal cord injuries and the major limitations of the existing animal models; and the outcome measures that have been developed to assess spinal cord injuries and the effectiveness of experimental therapies, including the development of imaging technologies.

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Spinal Cord Injury: Progress, Promise, and Priorities MOLECULAR, GENETIC, AND IN VITRO TOOLS Techniques have been developed that allow researchers to isolate and grow populations of neurons to investigate the effects of specific proteins and molecules on neuronal injury and repair. Neurons can be grown in isolation or with glial cells such as oligodendrocytes or Schwann cells to study the processes of axonal outgrowth and myelination. Investigators use molecular biology-based techniques, such as DNA or protein analysis, that can be used to easily visualize or analyze outcomes. Demonstrating the power of a cell culture experiment, the simple growth-cone turning assay led to the discovery that altering various molecules inside the growing axon regulates protein and cyclic nucleotide activities, which, in turn, can convert an axon’s response to a growth-inhibiting molecule from one of repulsion to one of attraction (Song et al., 1998). When this application is applied to regenerating axons in the rat spinal cord, investigators showed that the regrowth of transected neurons has the potential to be enhanced considerably (Neumann et al., 2002; Qiu et al., 2002). Furthermore, the recent elucidation of the signaling pathways responsible for this switch in response may lead to the discovery of a strategy for enhancing axon regeneration (Wen et al., 2004). Often, in vitro assays can be used in experiments with animal models, thus allowing researchers to verify and examine the effects detected in vitro to be evaluated in a more complex system. For example, chondroitin sulfate proteoglycans were found to inhibit neurite outgrowth in in vitro experiments (Snow et al., 1990). Analysis with animal models demonstrated that the levels of these proteoglycans are enhanced, or up-regulated, during central nervous system (CNS) injury (Snow et al., 1990) and led to the development of a strategy to break down these substances and promote the regrowth of axons in the intact rat spinal cord after an injury (Bradbury et al., 2002). Animal Models for Molecular and Genetic Studies Models consisting of multiple-transgenic animals have been developed to investigate molecular mechanisms and to identify the molecules critical for specific processes (Table 3-1). These models provide a better understanding of the genetic and molecular basis by which spinal cord circuits, specific neuronal subtypes, and synapses are formed (Shirasaki and Pfaff, 2002; Lanuza et al., 2004). For example, by studying the development of the nervous system of the fruit fly (Drosophila melanogaster), researchers have identified numerous molecules that can regulate the growth of the axon and the formation of neuronal connections (Vaessin et al., 1991; Kidd et al., 1998; Kraut et al., 2001; Jin, 2002). This information should provide

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Spinal Cord Injury: Progress, Promise, and Priorities TABLE 3-1 Animal Models Commonly Used to Identify Genes Involved in Axon Growth and Circuit Formation Animal Technique(s) Primary Utility Fruit fly Transgenic Identify and investigate molecular expression patterns; perform genetic experiments to identify the molecules involved in axon growth and guidance and the reformation of neuronal connections Worm Transgenic Identify and investigate molecular expression patterns and perform genetic experiments Fish Transgenic, transection, Examine motor control and the central pattern generator after transection of the spinal cord and investigate axonal regeneration models Mouse Transgenic, imaging Identify and investigate molecular expression patterns; perform genetic experiments to identify the molecules involved in axon growth and guidance and the reformation of neuronal connections; examine cellular and molecular basis of spinal cord circuits the insights needed to reconstruct effective circuits once axonal regeneration has been achieved. ANIMAL MODELS OF SPINAL CORD INJURY Animal models allow in-depth investigation of the anatomical and molecular changes that occur in response to a spinal cord injury at a level of detail that would not be possible or ethical in studies with humans. These insights are critical for the design and interpretation of the results of studies with humans. Without the knowledge gleaned from studies with animals, the spinal cord would remain the equivalent of a black box and therapies aimed at restoring function would be limited. For example, experiments with rodents demonstrated that the neurons in the spinal cord are able to regenerate after an injury (Richardson et al., 1980; Xu et al., 1995). Researchers have developed a variety of animal models that mimic

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Spinal Cord Injury: Progress, Promise, and Priorities different attributes associated with spinal cord injuries. Depending on the purpose of the study and the specific aspect of the injury to be investigated, researchers determine which animal model most closely replicates the injury in humans (Tables 3-2 and 3-3). In 2000, the International Spinal Research Trust published guidelines that describe four characteristics that are required for an optimal model of spinal cord injury (Ramer et al., 2000): The nature and the extent of the lesion should be precisely defined. If there is doubt about the extent of a lesion or whether axons have been spared, then interpretations of regeneration can be misleading. A histological method should be available to detect the growth of axons through the lesion. A method should be available to analyze the functional synaptic transmission beyond the lesion by measuring the electrical activity that neurons use to communicate with one another. A behavioral measure should be available that is capable of detecting restoration of known circuits. It is important to examine therapies in a system that best mimics the condition of the individual with a spinal cord injury. For example, therapies designed for individuals with chronic conditions should not be tested in animal models immediately after the animal has received the injury but should be tested only after the animal is in the chronic stage of the injury (Kwon et al., 2002a; Houle and Tessler, 2003; Kleitman, 2004). Further- TABLE 3-2 Value of Animal Models for Spinal Cord Injury Research Allows in-depth investigation of the anatomical changes that occur in response to an injury Regeneration of axonal tracts between the brain and the spinal cord can be studied in detail Individual components of the complex neural circuitry required for sensory perception and motor control can be examined Factors that influence DNA and proteins can be characterized Provides a means to examine the effects of specific genes Provides a tool to identify and test the efficacies of potential therapeutic agents and targets Identifies clinical end points that can be used to assess the efficacies of therapeutic agents

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Spinal Cord Injury: Progress, Promise, and Priorities TABLE 3-3 Criteria for Choosing an Ideal Animal Model Ability to match the behavioral complication to a morphology deficit Similarities and differences between the anatomy and cellular composition of the animal and human spinal cord Similarity of the whole injury process, including genetic changes and progression, to that observed in humans Similarities and differences between the timing of the stages of injury and life cycle in animals and humans Similarities and differences in the genetic backgrounds of the animal strains and species that may influence the response and recovery from a spinal cord injury Economics of the model, including the costs of care and feeding, and regulations   SOURCE: Croft, 2002. more, each type of spinal cord injury (Chapter 2) is different and presents its own set of challenges; therefore, each requires its own standard animal model that reliably mimics the complications experienced by individuals with that type of spinal cord injury. A number of animal models have been developed, including models that mimic compression, contusion, and transection (Table 3-4). Blunt contusion injuries account for 30 to 40 percent of all human spinal cord injuries (Hulsebosch, 2002); thus, the contusion model provides an important tool that researchers can use to examine the neuropathology of the injury and to test the efficacies of different therapeutic agents. In 1978, the clip compression technique was developed by researchers to simulate the continual pressure and displacement of the spinal cord common in spinal cord injuries, which is not reproduced in contusion injuries (Rivlin and Tator, 1978). This procedure has provided researchers with a great deal of information about the pathophysiology of the spinal cord during the acute stages of the injury; the timing, necessity, and effectiveness of releasing the pressure from the spinal cord; and potential therapies (Kwon et al., 2002b). To target and eliminate particular groups of neurons, methods that generate microlesions (Magavi et al., 2000) and that leave the vast majority of the nervous system intact have been developed. Using this strategy, the functional consequences that result from losing the nerve groups can be systematically examined. Researchers are determining the neuronal populations responsible for specific spinal cord injury deficits, including the root causes of chronic pain (Gorman et al., 2001).

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Spinal Cord Injury: Progress, Promise, and Priorities TABLE 3-4 Commonly Used Animal Models of Spinal Cord Injury Animal and Injury Modeled Primary Utility and Potential Issues Primate transection Test the safety and efficacies of therapies Determine the role of the central pattern generator in bipedal animals Ethical complications with the use of primates High cost of animal maintenance Limited number of animals that can be prepared for experimentation Spatial arrangement of the tracts differs from that in humans Cat contusion, transection Examine and define spinal cord circuitry and the central pattern generator Central pattern generator may have different amounts of brain regulation compared with that in humans Spatial arrangement of the tracts differs from that in humans Chromosomes and genes are organized differently from those in humans Mouse contusion, compression, transection, transgenic, microlesion formation Investigate molecular and anatomical changes that occur in response to injury; however, mice respond differently than humans to spinal cord injury Examine specific molecular targets for potential therapeutic targets Modify genes to test the effect on restoration or loss of function Difficult to assess upper extremity function Genetic variability in injury response, including scar formation Differences in scale size of spinal cord between mice and humans Spatial arrangement of the tracts differs between mice and humans Chromosomes and genes are organized differently from those in humans Rat contusion, compression, transection, microlesion formation Investigate molecular and anatomical changes that occur in response to injury Difficult to assess upper extremity function Differences in scale size of spinal cord in rats versus humans Chromosomes and genes are organized differently from those in humans NOTE: Contusion refers to a bruising of the spinal cord. Transection models are used to simulate lacerations to the spinal cord. Transgenic refers to modification of the animal’s genetic profile, which is done by deleting or modifying existing genes or introducing a novel gene.

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Spinal Cord Injury: Progress, Promise, and Priorities Issues Regarding Animal Models Mimicking Transection and Compression Injuries To make certain that the results from transection experiments are correctly interpreted and to minimize the variability in results, it is important that transection methods be standardized and that control animals be prepared at the same time that the experimental animals are treated. For example, to ensure that the recovery of function is due to axonal regeneration and not spared spinal cord circuitry, researchers must precisely perform transections of the spinal cord and must be sure that the axons projecting from the neurons are completely severed. If not all of the axons are severed, sparing and sprouting from uninjured axons become issues. It is important to note that damage to the dura mater as a result of a penetrating injury (including experimental transection) provides a route for the invasion of fibroblasts into the injury site (Zhang et al., 1996, 2004). Furthermore, in mice, there is extensive invasion of fibroblasts even without damage to the dura and the fibroblasts participate in the formation of a tissue matrix that is supportive for regeneration of at least some types of CNS axons. Following penetrating injuries, the potential contribution of fibroblasts (positive or negative) must be considered in evaluating experimental interventions to promote repair and functional recovery By virtue of the means by which compression injuries occur, there is a large amount of variability in the severities of spinal cord injuries. However, when initial compression studies are performed, it is important to be able to study a large population of animals that have the exact same initial injury characteristics before the experimental therapeutic intervention. Protocols have been developed to help minimize the variability in injury from animal to animal. Three impactors are widely accepted as standard methods for the delivery of contusion injuries to rodents: the Ohio State University (OSU) impactor, the Infinite Horizons device, and the Multicenter Animal Spinal Cord Injury Study (MASCIS) impactor (Bresnahan et al., 1987; Noyes, 1987; Kwo et al., 1989; Gruner, 1992; Young, 2002). Genetic Variability Between and Among Species Although it is important to test therapeutic interventions in animals before they can become established treatments in the clinic, genetic differences between animal species can potentially result in different responses to spinal cord injuries or treatments. For example, in response to injury, humans and rats develop a cavity in the spinal cord, but this does not occur in mice (although the precise cellular and molecular bases for this are not yet

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Spinal Cord Injury: Progress, Promise, and Priorities BOX 3-1 The Story of Nogo-Knockout Mice: Cooperation, Collaboration, and Genetic Variability Three groups of investigators recently used the gene-knockout strategy to examine whether Nogo, a potential inhibitor of axon growth (see Chapter 2), was responsible for preventing neuronal regeneration after an injury (Steward et al., 2003). Researchers coordinated their research efforts and published their findings in papers published in the same issue of the journal. Each group removed a specific part of a mouse’s chromosome that is responsible for Nogo, with the hypothesis that if Nogo is responsible for inhibiting neurons from growing, then its removal would facilitate regeneration after a spinal cord injury. However, the experiments found contradictory results. One study reported that the loss of Nogo increased the extent of neuronal regeneration, as predicted (but only in young mice), and the second study reported a more modest enhancement; however, the third group did not find any significant difference (Kim et al., 2003; Simonen et al., 2003; Zheng et al., 2003). The various results could have been due to differences in the ages and the genetic backgrounds of the mice, the strategy used to delete the Nogo gene, and the compensatory changes in other genes. In order to better understand the differences in these results, two of the groups have set up a collaboration to share their mice and perform their own analyses. This example demonstrates the value of genetic techniques, the importance of consistency in experimental design, the need to replicate experimental results, and the value of collaborative and collegial interactions between research groups. well understood). In amphibians, regeneration readily occurs directly through the glial scar. Different strains of the same animal species may respond differently to spinal cord trauma. For example, the nature and the extent of the secondary injury and wound healing vary in different strains of mice (Inman et al., 2002). Although these differences in responses between strains and species complicate comparison of the results of studies with different animal species, they may provide important insights about the specific genes that affect postinjury signaling cascades (Inman et al., 2002). Furthermore, the differences observed in experiments with the Nogo gene (Box 3-1) provide important lessons about the necessity to replicate experiments. Scale The human spinal cord is more than four times as long as the rat’s entire CNS (brain and spinal cord). Figure 3-1 demonstrates the difference in size between the entire CNS of a rat and the caudal end of a human spinal cord. A contusion or transection trauma in humans can affect upwards of 2 to 3 centimeters of the spinal cord, which is approximately 10

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Spinal Cord Injury: Progress, Promise, and Priorities FIGURE 3-1 Size discrepancy between the rat and the human spinal cords. The human spinal cord is more than four times as long as the entire CNS of the rat. (A) A caudal segment of the human spinal cord, including the cauda equina. The human cauda equina is approximately the same length as the entire CNS of a rat, which includes its brain. (B) The diameter of the human spinal cord is also much larger than that of the rat spinal cord. Twenty slices of a rat spinal cord can fit inside one slice of a human cord. SOURCE: Reprinted with permission, from Dobkin and Havton, 2004. Copyright 2004 from Annual Reviews. times the length of the 1 to 3 millimeters often affected by contusion injuries in rats (Metz et al., 2000). Consequently, regeneration of nerve fibers over a few vertebral segments in a rat—which can result in the restoration of function—is equivalent to only a fraction of the distance that is needed to restore function in humans (Dobkin and Havton, 2004). Furthermore, because neurons from both species demonstrate the same degree of spontaneous sprouting of their axons, approximately 2 millimeters (von Meyenburg et al., 1998), there are added complexities in promoting sufficient axon

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Spinal Cord Injury: Progress, Promise, and Priorities growth in humans (Dobkin and Havton, 2004). Although parts of the white matter of the human spinal cord are almost as large as the entire diameter of the rat spinal cord (Figure 3-1), there is no significant difference in the capacity for oligodendrocyte precursor cells to migrate to remyelinate axons in rats and humans. One of the issues regarding the differences in scale between smaller laboratory animals and humans that has been discussed is the extent to which testing is needed in primate models. Depending on the treatment, it may be advisable to examine the efficacies of some cell therapies in primates. However, there are also limitations in the use of non-human primates for mimicking human responses. For example, some types of monkeys have specific antibodies that can attack and inhibit the survival of human cells. Additionally, the bioavailability and metabolism of anti-rejection drugs in non-human primates and humans differ significantly. Therefore, rodents have frequently been used as the preferred model to study the efficacies of new immunosuppressive agents because of similarities in metabolism between rodents and humans. In addition, experiments are sometimes performed in rabbits and cats, which have larger spinal cords and are also less expensive and easier to maintain than primates. Furthermore, few tests have been developed to assess changes in spinal cord recovery in nonhuman primates. The committee believes that every therapy need not necessarily be tested in primates before clinical trials are performed with humans and that tests with primates be limited to those that will answer questions that are best explored only with non-human primate models. Next Steps The promise accorded by the methodical testing of therapies with animal models is beginning to pay off. Scientists have identified numerous inhibitory molecules and receptors that prevent the regeneration of neurons in the spinal cord and have clarified the pathways by which the inhibitory response can be modulated. Additional resources and tools are still needed in some areas, however. Animal models need to be developed for solid spinal cord injuries, as they account for a significant portion of human spinal cord injuries (Hulsebosch, 2002). Primate models of contusion injury are particularly needed, as well as standard animal models for cervical spinal cord injuries. Furthermore, there is no standard laboratory animal model that spinal cord injury researchers can use to examine fine motor control of the upper extremities or the loss of the sensory modality proprioception, which is responsible for limb position and immediately varying the degree of muscle contraction in response to external stimuli. When individuals with spinal cord injuries lose their proprioception, they are unable to move freely and interact comfort-

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Spinal Cord Injury: Progress, Promise, and Priorities ably with the external environment (see Box 5-1). Therefore, the development of a standard animal model that mimics the loss of proprioception will facilitate the development of therapies in a timely fashion. It is important that researchers use standardized animal models and that they use them consistently. The National Institute of Neurological Disorders and Stroke (NINDS), in recognition of the need to train researchers who work on spinal cord injuries, collaborated with Ohio State University to design a course that emphasizes competency in the technical approaches required for standard animal care and treatment and experimental design (Ohio State University, 2004). In addition, the University of California at Irvine has developed a similar course. These courses provide researchers with the opportunity to be trained to use the same standards for animal research. By training multiple researchers to use standard techniques, consistent animal injury models can be implemented. These models will increase the extent to which research results can be compared and improve the extent to which animal models can be used to predict clinical outcomes in humans. OUTCOME MEASURES USED TO ASSESS INJURY AND RECOVERY Because of the variations in the severity and the nature of the outcomes that individuals with spinal cord injuries experience, it is often difficult for health care professionals and researchers to assess the success of a particular intervention. Similarly, it is difficult for preclinical researchers to consistently assess progress in laboratory animal experiments and to determine the amount of progress, if any, that results from natural recovery, drug therapy, surgical intervention, or rehabilitation. Outcome Measures Used to Assess Spinal Cord Injury in Animal Models Tests developed to examine the recovery of function in laboratory animals have been designed primarily to examine motor function (Table 3-5; Appendix D). However, to accelerate the translation of research in other areas, including sexual function, bladder and bowel control, and chronic pain relief, standard tests need to be developed to assess experimental therapies for each of these major complications (Widerstrom-Noga and Turk, 2003). Researchers use a standard scale, the Basso, Beattie, and Bresnahan (BBB) scale, to assess the recovery of motor function in rats (Basso et al., 1995). The foundation of the BBB scale is the assessment of hind-limb movements in rats with spinal cord injuries. The 21-point BBB scale is sensitive enough that small gains in motor function are reflected in changes

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Spinal Cord Injury: Progress, Promise, and Priorities stimuli and mental tasks. It allows researchers and clinicians to study the changes in injured neuronal circuits. However, fMRI relies on the metabolic changes that occur in response to neural activity and the images obtained by fMRI are not a direct measure of neural activity. Therefore, caution should be placed on interpretation of the accuracies of the spatial maps generated by fMRI (Ugurbil et al., 2003). The National Institutes of Health has recommended that fMRI techniques be developed to assess the degree of loss and recovery of sensation in rodents with contusion injuries to their spinal cords (Hofstetter et al., 2003; NINDS, 2004). Radiologists use CT scans as a standard procedure to clarify areas of clinical concern (Youmans, 1996; AANS/CNS, 2002). Although MRI is better suited for analyzing the soft tissue of the spinal cord, the strength of using CT scans is in investigating the bone structure and detecting fractures of the vertebrae (Figure 3-2). Helical CT scans offer advantages over traditional radiology X-rays due to their speed in accruing the images and increased accuracy (4.5 minutes and 98.5 percent, respectively, for helical CT compared with 25 minutes and 43 percent, respectively, for X-rays). Therefore, in conjunction with MRI, CT scans provide useful tools for emergency clinicians (Nunez et al., 1994). FIGURE 3-2 MRI (A) and CT (B) of an injured spinal cord. Imaging of a spinal cord contusion injury by MRI and CT helps to reveal different aspects of the injury. The MRI image on the left reveals the soft spinal cord and bone, whereas the CT scan image on the right clearly delineates bone structures. SOURCE: Reprinted with permission, from AANS, 1999. Copyright 1999 from AANS.

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Spinal Cord Injury: Progress, Promise, and Priorities Unlike MRI, fMRI, and CT scans, PET scans detect and localize specific naturally occurring proteins; molecules, such as sugars and water; and other substances, such as neurotransmitters, which have been modified to emit radioactive energy. At present, PET scans are not commonly used in the clinic to assess spinal cord injuries. However, as discussed below, the technology has much potential to provide researchers and clinicians with a means by which to visualize changes in gene expression in the spinal cord. Next Steps: Future Imaging Technologies Imaging technologies provide clinicians with important tools to gauge the responses of patients to different therapies (Jacobs et al., 2003). The creation of sensitive assays that merge image-based technologies with biomarker research will allow investigators and clinicians to use specific tracers to localize molecular, genetic, and cellular processes in real time, thus providing further insight into the biological processes that affect the progression of the injury (Blasberg and Gelovani, 2002). As of January 2005, no clinical studies in the United States were specifically examining the use of imaging marker technologies for the study of spinal cord injuries. In comparison, markers are used to assess the state of MS and Alzheimer’s disease and imaging techniques are used to monitor the effects of different treatments for these conditions. For example, imaging assays are being developed to visualize specific neurotransmitter levels and to determine if they are involved in memory loss (Brown et al., 2003). The Future of Magnetic Resonance Technology In animals with syringomyelia, diffusion-weighted MRI, which is sensitive to the diffusion or random motion of water molecules in tissue, can detect cystic lesions in the gray matter of the spinal cord (Schwartz et al., 1999). The increased sensitivity offered by diffusion-weighted MRI will enable physicians to detect specific complications of spinal cord injuries sooner, thus increasing the potential for treatment. Magnetic resonance technology can be adapted to provide more than diagnostic information about the structural changes occurring in response to a spinal cord injury. In 2001, Bulte and colleagues used magnetic resonance to track oligodendrocyte stem cells that were prelabeled with super paramagnetic iron oxide nanocomposites, which are small beads invisible to the naked eye that can be detected by MR technology (Bulte et al., 1999, 2001). Using this approach, the investigators were able to track the real-time migration and integration of these oligodendrocyte stem cells for up to 6 weeks in the same animal, which is important for distinguishing the

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Spinal Cord Injury: Progress, Promise, and Priorities efficacies of endogenous cells versus those of the exogenous transplanted stem cells. The Future of PET Scans PET scan technology is being developed to inform clinicians about whether drugs can bind to the appropriate targets. For example, clinicians are using PET scans to determine if treatments are effective by looking at the uptake of glucose, which tumors need to nourish their growth (Van den Abbeele and Badawi, 2002; Pollack, 2004). These effects can be observed before structural changes in the tumor can be detected. Two caveats about the use of PET scans must be kept in mind. First, current technology does not have enough resolution to allow complete visualization through the entire diameter of the spinal cord. Furthermore, the current spatial resolution of commercial PET scanners is 4 mm but 2.5 mm resolution has been achieved in research instruments that use motion compensation. Second, information obtained from PET scans is based on metabolic events that correlate to neural activity and may not directly correspond to the location where the changes in activity are occurring. Therefore, the images generated by PET scans could be misleading because they may not accurately represent the spatial specificities of the changes (Ugurbil et al., 2003). However, refinements to PET scans could provide important information about the cellular states of the injury, such as gene activation or suppression in response to the injury; this would provide physicians with the ability to quantify responses to different spinal cord injury treatments (Brooks et al., 2003) and to identify functional changes before the onset of structural changes identifiable by MRI (National PET Scan Management, LLC, 2004). PET ligands have been developed that can detect glucose metabolism, inflammation, and receptor abundance, including agents that track the N-methyl-D-aspartate (NMDA) receptor activity and proteases. PET measures very different process than does MRI whose spatial resolution is superior. However, PET contrast resolution for identification of proteins can be hundreds of times greater than MRI depending on the target. The potentials of PET for assessing the severity of injury and the responses to therapy await application of high resolution systems with recently developed radiopharmaceuticals. Tracking Recovery with PET and Magnetic Resonance Improvements to PET and MR technologies enable investigators to visualize the molecular signatures of damage and repair to the CNS. In an attempt to examine the activities of specific neuronal circuits, imaging markers that mimic neurotransmitters and receptors that are nonradioactive are

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Spinal Cord Injury: Progress, Promise, and Priorities being created, including the iron analog annexin V (Schellenberger et al., 2002), the fluorescent marker Cy5.5 (Petrovsky et al., 2003), and markers that do not become active until they reach their target. Future modification and adaptation of these technologies could be used to examine specific stages of regeneration, including those designed to detect neurite outgrowth, astrocyte scarring, oligodendrocyte myelination, and immunological response. Transgenic Animals: Following the Labeled Cell At present it is difficult to follow the path of cell transplants (such as stem cells, Schwann cells, and olfactory ensheathing cells) in the living spinal cord; therefore, it is difficult to draw conclusions about the efficacy of an experiment with such cells. Continued advancement of imaging techniques will provide a mechanism by which investigators and clinicians can assess the integration of grafted tissue or cells into the preexisting neuronal network or monitor the response to gene therapy by tracking the transgene location. Transgenic animal models have thus been developed. Specific populations of cells in these animals are genetically engineered to be fluorescent or to emit a fluorescent signal when they are functionally activated. Such approaches, which use two-photon confocal imaging to detect the signal, can be directly applied to spinal cord preparations in vitro and administered to intact mice and rats. With improvements in the current technology, the use and improvement of near-infrared markers might also provide researchers with a means to monitor the progression of a spinal cord injury and recovery in laboratory animals. Multidisciplinary Research and Bringing Molecular Imaging to the Clinic The promise of molecular imaging technologies can be realized only if the technologies can be successfully transferred to the clinical setting. The transfer of these technologies will require cross-disciplinary collaborations and multidisciplinary research efforts among molecular and cellular biologists, imaging scientists, nanotechnologists, and clinicians. A review article by Massoud and Gambhir (2003) identified the following goals for the transfer of molecular imaging technologies from the research laboratory to the clinic: develop noninvasive in vivo imaging methods that detect specific cellular and molecular processes, such as gene expression and protein-protein interactions; monitor multiple molecular events in concert; monitor the trafficking and targeting of cells;

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Spinal Cord Injury: Progress, Promise, and Priorities optimize drug and gene therapies; image drug effects at the molecular and cellular levels; and assess the molecular pathology of disease progression. Achieving these goals and translating those achievements into reliable clinical technologies will be critical steps toward the treatment and diagnosis of spinal cord injuries at the molecular level. To achieve these objectives, continued advances need to be made to overcome the challenges of biocompatibility, probe delivery, and high-resolution signal detection (Mahmood and Weissleder, 2002). Cross-disciplinary collaboration and multidisciplinary research is needed to bring together molecular and cellular biologists, imaging scientists, nanotechnologists, and clinicians to reach these goals (Blasberg and Gelovani, 2002). Many of the imaging techniques used to examine the CNS were designed to visualize brain tumors or to assess Alzheimer’s disease, Parkinson’s disease, and MS. These resources and technologies can be applied or can provide models for spinal cord injury research. For instance, investigators are examining the utility of using multiphoton imaging techniques to monitor the progression of senile plaques in mice that model Alzheimer’s disease (Christie et al., 2001). This technology could also be modified to assess and monitor the progression of the glial scar formation that results from spinal cord injuries. The cancer research field not only has led the way in developing technologies but also has helped to establish research centers that have been critical in creating a means for translating imaging technologies into the clinic. In particular, the National Cancer Institute has developed two programs: the Small Animal Imaging Resources Program (SAIRP) and the In Vivo Cellular and Molecular Imaging Centers (ICMIC) Program. These programs, along with support mechanisms sponsored by the National Institute of Biomedical Imaging and Engineering, provide mechanisms and model systems that can be used to promote the cooperative development of new imaging systems for spinal cord injury research and treatment. RECOMMENDATIONS Recommendation 3.1: Increase Training Efforts on Standardized Research Tools and Techniques Spinal cord injury researchers should receive training in the use of standardized animal models and evaluation techniques. Pre- and postdoctoral fellowship training programs focused on spinal cord injury research should require participation in courses designed to train investigators on the appropriate use of the available tools and techniques.

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Spinal Cord Injury: Progress, Promise, and Priorities Recommendation 3.2: Improve and Standardize Research Tools and Assessment Techniques Preclinical research tools and animal models should be developed and refined to examine spinal cord injury progression and repair and assess the effectiveness of therapeutic interventions. These preclinical tools and assessment protocols should be standardized for each type and each stage of spinal cord injury. Particular emphasis should be placed on: improving imaging technologies to allow real-time assessment of the current state and progression of the injury; identifying biomarkers that can be used to monitor the progression of the injury and recovery; developing additional animal models to explore the progression of spinal cord injury and repair; establishing standardized sets of functional outcome measures for the evaluation of experimental therapies for each type and each stage of spinal cord injury in animal models; and enhancing functional assessment techniques to examine motor function as well as secondary complications, including pain and depression of the immune system. REFERENCES AANS (American Association of Neurological Surgeons). 1999. Spinal Cord. [Online]. Available: http://www.neurosurgerytoday.org/what/patient_e/spinal.asp [accessed January 25, 2005]. AANS/CNS (American Association of Neurological Surgeons/Congress of Neurological Surgeons). 2002. Radiographic assessment of the cervical spine in symptomatic trauma patients. Neurosurgery 50(3 Suppl): S36-43. Bareyre FM, Schwab ME. 2003. Inflammation, degeneration and regeneration in the injured spinal cord: Insights from DNA microarrays. Trends in Neurosciences 26(10): 555-563. Bareyre FM, Haudenschild B, Schwab ME. 2002. Long-lasting sprouting and gene expression changes induced by the monoclonal antibody IN-1 in the adult spinal cord. Journal of Neuroscience 22(16): 7097-7110. Basso DM, Beattie MS, Bresnahan JC. 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. Journal of Neurotrauma 12(1): 1-21. Blasberg RG, Gelovani J. 2002. Molecular-genetic imaging: A nuclear medicine-based perspective. Molecular Imaging: Official Journal of the Society for Molecular Imaging 1(3): 280-300. Bradbury EJ, Moon LD, Popat RJ, King VR, Bennett GS, Patel PN, Fawcett JW, McMahon SB. 2002. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416(6881): 636-640. Bregman BS, McAtee M, Dai HN, Kuhn PL. 1997. Neurotrophic factors increase axonal growth after spinal cord injury and transplantation in the adult rat. Experimental Neurology 148(2): 475-494.

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