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P&F Grant Awards

Year 6

Grant # 12

In Vivo Contrast-enhanced MicroCT Imaging of the Murine Intervertebral Disc

PI: Simon Tang, PhD


Specific Aims

Degeneration of the intervertebral disc (IVD) is a leading contributor towards back pain, an epidemic that costs billions of dollars in the US [1]. The IVD consists of a proteoglycan(PG)-rich nucleus pulposus (NP) surrounded by a collagenous annulus fibrosus (AF) [2] that together provide support and transmit complex loads [3,4]. The IVD degenerative cascade involves a multifactorial progression of biological, biochemical, and structural changes that lead to the collapse of the disc structure and to compromised mechanical function [3-5]. Despite its significant public health impact, the pathophysiology of disc degeneration remains unclear.

The lack of high-resolution imaging tools to monitor disease progression is one of the key hurdles in understanding the pathophysiology of IVD degeneration. Although magnetic resonance imaging (MRI) is commonly used to quantify IVD morphological and compositional changes in vivo for humans and large animal models [6], the relatively low resolution of MRI (hundreds of microns to millimeters) limits its sensitivity towards sub-millimeter and nuanced changes that may occur with the initiation of degeneration. Higher resolution imaging methods may improve the detection fidelity of the early degenerative process and the ability to leverage rodent models to understand the genetic and mechanobiologic interactions in intervertebral disc homeostasis.

Recent investigations have shown that micro-CT in the presence of contrast agents improves the discrimination and enables the 3D evaluation of unmineralized soft tissues. Ionic contrast agents such as ioxaglate (Hexabrix) proportionally binds to the negatively charged glycosaminoglycans (GAGs) in soft tissue such as cartilage [7,8], and the tissues are revealed by the differential uptake of contrast agent as detected by micro-CT. While ionic contrast agents are highly specific for charged tissues, they tend to be hydrophobic [9] and increase the propensity for in vivo endothelial injury [10] and renal nephropathy [11,12]. Nonionic, low osmolar, and hydrophilic contrast agents such as Ioversol (OptiRay) significantly reduce the complications [9,11] without compromising radiodensity, making them more suitable for in vivo imaging. No published studies to date have examined the effectiveness of these nonionic, hydrophilic agents for contrast-enhanced microCT imaging of the intervertebral disc in rodents.

Our laboratory recently explored the use of Ioversol for contrast-enhanced micro-CT imaging of rodent intervertebral discs. Taking advantage of the high-resolution of microCT (~10 microns) and the increased tissue hydration of the NP over the AF, Ioversol proves to be ideal for nondestructive 3D visualization of the IVD. Moreover, we have developed a set of parameters to distinguish the NP and the AF, and to quantify the intervertebral disc structure and proteoglycan content of the disc (NP Volume Fraction, NP Intensity Fraction, etc). Our preliminary ex vivo studies quantitatively captured the nuanced changes in tissue- and structurallevel changes due to trypsin-induced degeneration of the IVD [13,14]. Building on this work, we will investigate the in vivo longitudinal imaging of murine IVD using contrast-enhanced microCT with the overall hypothesis that the IVD degenerative cascade initiates dose-dependently with enzymatic insult.

SA 1: Compare and optimize the timing and efficacy of Ioversol for in vivo intervertebral disc structural imaging in the rat. We will directly deliver the contrast agent to the caudal intervertebral disc of Sprague Dawley rats and image these discs in vivo over 48 hours to determine the effects of local uptake and clearance of Ioversol on x-ray attenuation. Delivery of Ioversol will be achieved through direct injection to the caudal disc of interest, and adjacent discs will serve as internal controls.

SA 2: Longitudinally monitor spatial-temporal structural and biochemical changes with trypsin-mediated intervertebral disc degeneration and subsequent adaptation over 3 weeks. Trypsin, a serine protease that catalyzes the hydrolysis of peptide bonds such as those on sulfated GAGs, has been used to chemically degenerate the IVD to induce herniation [15] and is commonly used for the ex vivo validation of IVD MRI techniques [13,14,16,17], yet the spatial-temporal progression of IVD degeneration and adaptation remain relatively unknown. Using our developed technique to monitor the effects of low-, medium-, and high- doses of trypsin injected into the IVD, we will capture structural and compositional changes locally within the IVD, giving insight to the degenerative and regenerative response over time due to varying degrees of nucleolytic insult.

The research proposed here, led by a junior investigator, will heavily utilize the Histology and the Structure/Strength cores of the Musculoskeletal Research Center. The successful completion of these aims will validate a powerful new tool for analyzing the progression and treatment of intervertebral disc degeneration in small animal models, as well as provide critical preliminary data towards future NIH submissions.