From Bench to Bedside to Bench: Translation of Animal Models to Clinical Practice to Animal Models

Transcription

1 From Bench to Bedside to Bench: Translation of Animal Models to Clinical Practice to Animal Models Traumatic Brain Injury: MRI Assessment of Cerebral Blood Flow and Macrophage Accumulation in Mouse Models for Traumatic Brain Injury Lesley M Foley Pittsburgh NMR Center for Biomedical Research Mellon Institute Carnegie Mellon University 4400 Fifth Ave Pittsburgh, PA, USA Traumatic Brain Injury (TBI) According to the Centers for Disease Control and PreventionTraumatic brain approximately 1.4 million people sustain a traumatic brain injury annually and is a serious public health problem in the United States. Each year, traumatic brain injuries contribute to a substantial number of deaths and cases of permanent disability. Traumatic brain injury (TBI) is a complex process that is heterogeneous which can differ in the type of injury, and the distribution and mechanisms of damage. TBI occurs in overlapping phases from primary through to secondary damage. Primary damage can result from mechanical forces such as direct injury to the scalp, skull fracture, brain contusion from movement against rough surfaces of the skull and/or a contracoup contusion on the opposite side of the impact, and subdural hematomas, just to name a few (Gennarelli and Graham, 2005). Secondary injury mechanisms include hypoxia, ischemia, hypotension, swelling and edema (Gennarelli and Graham, 2005). The purpose of experimental models of TBI is to replicate pathological components of human TBI, therefore injury models must be capable of producing trauma with a wide range of severity. The most commonly used experimental models of TBI are the fluid percussion injury model (FPI), the controlled cortical impact model (CCI), and the impact acceleration model, all of these models have been used over a wide range of species. In the FPI model the injury is produced by the application of a fluid pressure pulse which rapidly strikes the intact dura and causes deformation of the underlying brain, the severity depends on the pressure pulse (Dixon et al., 1987). An FPI injury can be applied midline (Dixon et al., 1987) or laterally (McIntosh et al., 1989). Midline FPI cause bilateral cortical alterations with herniation to the brainstem, whilst lateral FPI inflicts unilateral cortical damage. The physiological responses include reduced cerebral blood flow (CBF), changes in blood pressure, reduced cerebral perfusion pressure (CPP), etc. The CCI Model employs a compressed air driven piston to impart mechanical energy to the intact dura (Lighthall, 1988; Dixon et al., 1991). The velocity and depth of the impactor and be varied depending on the type of injury required. It is able to Proc. Intl. Soc. Mag. Reson. Med. 18 (2010)

2 reproduce changes reported in clinical head injury such as edema, increased intracranial pressure (ICP), decreased CBF, coma, etc. The impact acceleration model (Marmarou et al., 1994) uses a device that consists of brass weights falling freely via gravity from a designated height within a tube. The animals skull has a steel disc fixed to and are then placed onto foam that allows defined movement of the animals head. The injury is created by the impact of the brass weights dropping onto the steel disc. This model produces coma, widespread axonal swelling, respiratory depression, decreased CBF, elevated ICP, etc. MRI Assessment of Macrophage Accumulation in a Mouse Model of TBI Cell specific imaging has become an important field in MRI. The increased development of superparamagnetic iron oxide particles as contrast agents has allowed this field to grow. Phagocytic monocytes play an important role with phagocytic, antigen-presenting and secretory functions, thus mapping their accumulation with magnetic resonance microscopy (MRM) and tracking the response of these cells with MRI could lead to greater understanding of the pathophysiology of numerous diseases. To develop effective therapies for the treatment of TBI a thorough appreciation of the role of macrophages could be essential. Recently, it has been shown that micron-sized paramagnetic iron oxide (MPIO) particles can label cells for in vivo detection by MRI (Hinds et al., 2003; Shapiro et al., 2004). Macrophages readily internalize these particles, therefore it is possible to label these cells directly in vivo (Wu et al., 2006). An advantage with the larger sized particles is that single cells may be visualized with MRI in vivo because single macrophages are able to take up a large amount of iron (Shapiro et al., 2006 and Wu et al., 2006). We used MPIO particles to detect the response of macrophages using MRM and in vivo MRI after experimentally induced TBI produced by controlled cortical impact (CCI) in a mouse model. Male C57BL/6J mice between 11 and 15 weeks of age were anesthetized with 2% isoflurane via nose cone. Mice were placed in a stereotaxic holder and a temperature probe was inserted through a burr hole into the left frontal cortex and the left parietal bone was removed for trauma. Once brain temperature reached 37 C ± 0.5 C and was maintained at this temperature for 5 min, a vertically directed CCI was delivered at a speed of 5.0 m/sec and a depth of 1.0 mm. After injury the bone flap was replaced, sealed with dental cement and the incision closed. This level of CCI produces a moderate to severe injury with damage to the underlying hippocampus and thalamus. The MPIO particles were purchased from Bangs Laboratories, Fishers, IN. They are 0.9 µm superparamagnetic styrene-divinyl benzene inert polymer microspheres that contain a magnetite core and a fluorescein-5-isothiocyanate dye (Dragon Green fluorescent probe) contained within the cross-linked polymer sphere. MPIO was administered to the mice as an intravenous bolus dose of 4.5 mg Fe/kg body weight, through a catheter placed in the femoral vein.

3 Figure 1. Visualization of macrophage accumulation by high-resolution magnetic resonance microscopy (MRM) of representative excised brains at 11.7 Tesla. Panel A depicts naïve mouse with no contrast injection. Panel B depicts a low level of randomly localized areas of punctuate hypointensity (arrows and arrowheads) representing MPIO particles from a naïve mouse that was injected 24 h earlier with MPIO particles. Panel C depicts a small number of particles in brain from a mouse that was injected with MPIO particles at 24 h after CCI and imaged at 48 h after CCI. Panel D depicts more robust contusional and peri-contusional accumulation of particles in a mouse that was injected with MPIO particles at 48 h after CCI and imaged at 72 h after CCI. Panel E depicts a smaller number of particles in a mouse that was injected with MPIO particles at 6 days before CCI and imaged at 48 h after CCI. MPIO labeled cells are best visualized with MRM due to the increase in resolution. Discrete black spots can be seen in the MRM images from the excised brains in mice that were injected with MPIO when compared with naïve (Figure 1). A low level of random particles were seen in naïve brains in mice that were injected with MPIO and imaged 24 h later (Figure 1B). Mice imaged at 48 h after CCI and 24 h after MPIO injection revealed a few particles in the injured hemisphere in peri-contusional regions (Figure 1C). Maximal particle accumulation in and around the contusion site was seen in mice imaged at 72 h after CCI and 24 h after MPIO particle injection (Figure 1D). When MPIO particles were injected 6 days before CCI and mice imaged 48 h after CCI, particles were still detected in the contusion and peri-contusional sites (Figure 1E) suggesting that labeled macrophages were still present at 6-8 days after injection and were able to travel to the trauma site. This supports the argument that the hypointense signal imaged in our studies represents labeled macrophages, rather than a nonspecific accumulation of MPIO particles traversing an acutely injured blood brain barrier (BBB).

4 This is further supported by the extremely short half-life of these particles in blood which has been found to be in the order of minutes in rats (Ye et al., 2008). It also supports the ability of labeled macrophages to be available to accumulate to an injury site, even when labeled 6 days prior to the insult. References Dixon, C. E., Clifton, G. L., Lighthall, J. W., Yaghmai, A. and Hayes, R. L. (1991) A controlled cortical impact model of traumatic brain injury in the rat. J. Neurosci. Methods 39, Dixon, C. E., Lyeth, B. G. and Povlishock, J. T. (1987) A fluid percussion model of experimental brain injury in the rat: Neurological, physiological, and histopathological characteristics. J. Neurosurg. 67, Gennarelli, T. A. and Graham, D. I. (2005) Acceleration induced head injury in the monkey. I. The model, its mechanical and physiological correlates. Acta Neuropathol. Suppl. 7, Hinds, K. A., Hill, J. M., Shapiro, E. M., Laukkanen, M. O., Silva, A. C., Combs, C. A., Varney, T. R., Balaban, R. S., Koretsky, A. P., and Dunbar, C. E. (2003) Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells. Blood 102, Lighthall, J. W. (1988) Controlled cortical impact: a new experimental brain injury model. J. Neurotrauma 5, Marmarou, A., Foda, M. A. A.-E., van den Brink, W., Campbell, J., Kita, H. and Demetiadou, K. (1994) A new model of diffuse brain injury in rats. Part 1: Pathophysiology and biomechanics. J. Neurosurg. 80, McIntosh, T. K., Vink, R., Noble, L., Yamakami, I., Fernyak, S. and Faden A. I. (1989) Traumatic brain injury in the rat: characterization of a lateral fluid percussion model. Neuroscience 28, Shapiro, E. M., Sharer, K., Skrtic, S., and Koretsky, A. P. (2006) In vivo detection of single cells by MRI. Magn. Reson. Med. 55, Shapiro, E. M., Skrtic, S., Sharer, K., Hill, J. M., Dunbar, C. E., and Koretsky, A. P. (2004) MRI detection of single particles for cellular imaging. Proc. Natl. Acad. Sci. USA. 101, Wu, Y. L., Ye, Q., Foley, L. M., Hitchens, T. K., Sato, K., Williams, J. B., and Ho, C. (2006) In situ labeling of immune cells with iron oxide particles: an approach to detect organ rejection by cellular MRI. Proc. Natl. Acad. Sci. USA. 103,

5 Ye, Q., Wu, Y. L., Foley, L. M., Hitchens, T. K., Eytan, D. F., Shirwan, H., and Ho, C. (2008) Longitudinal tracking of recipient macrophages in a rat chronic cardiac allograft rejection model with non-invasive MRI using micrometer-sized paramagnetic iron oxide particles. Circulation 118,