Spinal cord injury (SCI) is a devastating neurological disorder affecting approximately 12,000 individuals in the United States each year¹. Secondary injury, which occurs hours to months following initial primary traumatic insult, contributes to metabolic stress and progressive tissue damage, and serves as the main target for therapeutic intervention. Knowledge of spinal cord perfusion is important not only for the understanding of underlying physiology, but also for monitoring treatment. Clinical monitoring of spinal cord perfusion and secondary injury relies on secondary measures such as blood pressure and neurologic exam. Current non-invasive imaging methods for directly assessing spinal cord perfusion are significantly hampered by limited spatial resolution, physiologic motion and magnetic field inhomogeneity related to the bony spine². Dissolution dynamic nuclear polarization (DNP) enables the acquisition of ¹³C MR data with a huge gain in sensitivity³. Recent studies using metabolically inactive ¹³C-labeled hyperpolarized (HP) compounds have demonstrated the feasibility of using this technique as a new perfusion MR method in preclinical cancer models⁴. The purpose of this study was to explore the feasibility of using HP ¹³C MRI with [¹³C]t-butanol and [¹³C,¹⁵N₂]urea for evaluating in vivo perfusion of spinal cord in rodents as a first step in preparing for an in vivo study of traumatic injury. To our knowledge this is the first study that has demonstrated the acquisition of HP ¹³C perfusion imaging from the spinal cord.Healthy female Long-Evans rats (n=3) were included in this study. All experiments were performed using a GE 3T scanner with a custom-designed ¹H/¹³C-coil. The cervical lordosis was straightened with padding under the ventral neck to minimize partial voluming with non-spinal tissue (Figure 1D). 95μL [¹³C]t-butanol mixed with glycerol (50:50 by weight), 15mM trityl radical and 1.0mM Dotarem were polarized using a HyperSense^® polarizer and dissolved in 4.5mL phosphate-buffered saline (PBS). 135μL [¹³C,¹⁵N₂]urea mixed with glycerol (6M), 15mM trityl radical and 1.5mM Gd-DOTA were polarized using a SpinLab polarizer and dissolved in a mixture of 750μL PBS and ~13.5g of Tris-buffered aqueous solution. 2.7 mL HP [¹³C]t-butanol (100mM, pH=7.5) solution was injected through tail vein over 10s. At 7s from the start of injection, symmetric, ramp-sampled ¹³C EPI data were acquired every 4s for a total of 16 datasets (TR=1s, FOV=64×64mm, matrix=32×32, seven 6mm-slice, 30°-flip)⁵. Following t-butanol data acquisition, 2.7mL HP [¹³C,¹⁵N₂]urea (80mM, pH=7.5) solution was injected and data acquired using the same method. T-butanol and urea magnitude signal were overlaid on T2-weighted FSE images and area-under-the-curve (AUC), maximum peak signal and time-to-maximum-signal were calculated for pixels corresponding to spine, supratentorial brain and blood vessels. In addition, preliminary ASL data was acquired using a standard clinical ASL sequence (TR/TE=5686/10.1ms, FOV=14cm, points=512, spiral readout with 8 arms, NEX=16, ten 6mm-slice).Figure 1 shows examples of dynamic ¹³C t-butanol and urea data from regions around cervical spinal cord at C3-C4 vertebral level (Fig1A), supratentorial brain (Fig1B) and with high vascular signal (Fig1C). Slice locations are indicated on sagittal scout image (Fig1D). T-butanol and urea imaging revealed informative differences in the distribution in different tissues. T-butanol rapidly crossed the blood-brain barrier (BBB) and was freely diffusible in spinal cord and brain (Fig1A,1B). Unlike t-butanol, signal from urea predominantly came from vasculature (Fig1C) and was negligible in spine and brain tissue due to its limited ability to cross BBB⁶. The maximum SNR of t-butanol and urea in the spine was 16 ± 5 and 5 ± 0.4 (mean±SD), respectively, and the AUC 97 ± 30 and 26 ± 2 (mean±SD), respectively (Table1). The representative time courses of t-butanol and urea signal were plotted over time for different tissues (Fig2). The t-butanol and urea from blood vessels reached their maximum earlier than those from brain and spinal cord (Table1). The map of AUC for HP perfusion agents and preliminary ASL data from regions with different tissues (Fig3) indicate that this novel imaging method may provide complementary information to study perfusion in various tissue types. With the high spatial resolution of this HP ¹³C data (2x2mm in-plane pixel), it should be possible to visualize and compare hemicontusion lesions with a contra-lateral hemi-cord. The distinct physiological characteristics of t-butanol and urea may be used to estimate the perfusion and permeability of different tissue compartments in future studies with injury models⁴. We have demonstrated the feasibility of using HP ¹³C MRI with [¹³C]t-butanol and [¹³C,¹⁵N₂]urea for assessing in vivo perfusion in the cervical spine of uninjured rats. The results suggest that this technique may provide a unique non-invasive imaging tool that is able to monitor hemodynamic processes underlying spinal cord injury.