In vivo contrast‐enhanced microCT for the monitoring of mouse thoracic, lumbar, and coccygeal intervertebral discs

Abstract Mouse models are often used for studies of intervertebral disc (IVD) homeostasis and degeneration, yet the relatively small size of the IVD poses challenges for noninvasive, longitudinal imaging modalities. The recently developed contrast‐enhanced microCT (CEμCT) using Ioversol has been successful in detecting degenerative changes in the murine IVD ex vivo at the micrometer scale. Further leveraging the superior biocompatibility of Ioversol as a contrast agent, we demonstrate the in vivo use of this CEμCT technique to examine IVDs at multiple spinal sites. Ioversol was administered via tail vein injection (TVI) in growing and adult male FVB/NJ mice (n = 5 /group). The animals were anesthetized and underwent in vivo micro‐computed tomographic (microCT) at the coccygeal (CC5/CC6), lumbar (L5/6), and thoracic (T12/T13) IVDs. TVI of Ioversol was well‐tolerated by all animals. As Ioversol filtered through the kidneys and accumulated in the bladder, the attenuations of the mouse bladder and kidneys increased due to the high molecular weight of Ioversol, confirming that the Ioversol is biological available. Average IVD attenuations increased 3%‐15% following TVI (ANOVA; P < .01). The presence of Ioversol in the IVD combined with high‐resolution microCT allow for nondestructive visualization of structural features of the IVD. These results demonstrate CEμCT with Ioversol as a viable strategy for the in vivo monitoring of multiple mouse IVDs during degeneration, disease, and injury.

superior biocompatibility of Ioversol as a contrast agent, we demonstrate the in vivo use of this CEμCT technique to examine IVDs at multiple spinal sites. Ioversol was administered via tail vein injection (TVI) in growing and adult male FVB/NJ mice (n = 5 /group). The animals were anesthetized and underwent in vivo microcomputed tomographic (microCT) at the coccygeal (CC5/CC6), lumbar (L5/6), and thoracic (T12/T13) IVDs. TVI of Ioversol was well-tolerated by all animals. As Ioversol filtered through the kidneys and accumulated in the bladder, the attenuations of the mouse bladder and kidneys increased due to the high molecular weight of Ioversol, confirming that the Ioversol is biological available. Average IVD attenuations increased 3%-15% following TVI (ANOVA; P < .01). The presence of Ioversol in the IVD combined with high-resolution microCT allow for nondestructive visualization of structural features of the IVD. These results demonstrate CEμCT with Ioversol as a viable strategy for the in vivo monitoring of multiple mouse IVDs during degeneration, disease, and injury.

K E Y W O R D S
aging, Contrast-enhanced microCT, intervertebral disc, mouse model

| INTRODUCTION
Low back pain is one of the most common causes of disability. 1 With intervertebral disc (IVD) degeneration being a leading contributor to the development of low back pain, there is a need for a better understanding of the biological and structural changes of the IVD that occur with the disease and injury. 2 Mouse models are useful for studying the molecular mechanisms of IVD biology because of the number of genetically modified models and established techniques to induce degeneration. 3 However, resolving the micrometer-scale structural features of the mouse IVD remains a significant challenge even with state-of-the-art small animal microMRI technologies. 4 Micro-computed tomographic (microCT) techniques have outstanding spatial resolution for the volumetric imaging of mineralized tissues in rodents. 5 However, since nonmineralized tissues do not attenuate X-Rays, a contrast agent is required to differentiate the soft tissue of interest. Although ionic and heavy-metal and ionic contrastagents have shown to improve the X-ray attenuation of IVDs for ex vivo imaging 6,7 , the nonionic, hydrophilic, and highly biocompatible nature of Ioversol make it particularly suited for in vivo use. 8 Ioversol has been used clinically as a diagnostic radiographic contrast agent.
With a high molecular weight, 807.115 g/mol, Ioversol reduces the xrays that can pass through the tissue and increases the attenuation of blood vessels, organs, and other nonmineralized tissues. We have previously reported on a contrast-enhanced microCT (CEμCT) technique using Ioversol to monitor and quantify IVD degeneration in vitro and ex vivo. 9,10 Extending this work further, the objective of this study is to explore and validate the in vivo use of CEμCT technique using Ioversol to examine intervertebral discs at multiple spinal sites using growing and adult FVB/NJ mice.

| Animals
All procedures were performed with the approval of Washington University School of Medicine Institutional Animal Care and Use Committee. Male FVBN/J mice at two age groups, 3-(growing) and 8-(adult) month old, were used in this study (n = 5 / group). Wild-type mice were selected for this study to examine the diffusion of Ioversol into the IVD under healthy conditions. Mice were kept in standard husbandry conditions with 12-hour light/dark cycles as described by Jackson Labs (https://www.jax.org/jax-mice-and-services/customersupport/technical-support/breeding-and-husbandry-support/mouseroom-conditions). Mice were injected with 8 mL/kg of 350 mg/mL of Ioversol (OptiRay 350; Guerbet, St. Louis, Missouri) which reflects veterinary recommendations for a mouse at the highest available concentration of Ioversol. Mice were placed in a restrainer on a heat pad and lidocaine was applied to the tail. A 29G syringe was used to inject Ioversol into the lateral tail vein of the mouse. Mice were then anesthetized by isoflurane prior to microCT imaging.

| MicroCT imaging
MicroCT imaging was conducted on a VivaCT40 system (Scanco Medical, CH) at 10 μm voxel size (45 kVp, 177 uA, 116 ms integration time). Our preliminary studies showed that at least 20 minutes is required for TVI-administration of Ioversol to diffuse into the intradiscal space to increase the attenuation of the tissue. We

| Image processing
The raw data was exported to DICOM for further analysis using a cus-   Figure 1C), with the average increase ranging from 3% to 15% (P < .01; Figure 2A,D). Moreover, there is a reduction in the number of low attenuating voxels. Taken together, the Ioversol delivered by TVI was biologically available, and the Ioversol was transported to the IVD where it was preferentially taken up compared to the surrounding soft tissues.

| Statistics
In order to explore the time-dependence of Ioversol retention in the IVD, we monitored each intervertebral disc at three timepoints as technically allowed by the microCT system. Mean attenuation varied by less than 5% between the three post-TVI scans (data not shown). Increased attenuation following injection with Ioversol was observed across all levels and ages (Figure 2A

| DISCUSSION
To date, this is the first study to describe the in vivo use of CEμCT for the imaging of the mouse intervertebral disc. This approach was previously demonstrated in several ex vivo rodent models of IVD degeneration, and it achieved superior spatial resolution compared with microMRI. 9,10 By delivering Ioversol through tail-vein injection at a physiologically compatible dose, Ioversol was biologically available and accumulated preferentially in the IVD to increase the attenuation of all the scanned IVDs. We obtained disc height and volume measures of the IVDs expected from the aging mouse IVD. 6,11,12 The TVIs were well-tolerated by all mice with no adverse consequences, suggesting that the technique would be suitable for longitudinal monitoring. However, it is worth noting that Ioversol is reported due to radiation. 13,14 CEμCT will be a valuable strategy for in vivo monitoring of IVD injury and degeneration across multiple levels in mouse models.