Postnatal Development of the Murine Notochord Remnants Quantified by High-resolution Contrast-enhanced MicroCT

Abstract

The notochord gives rise to spinal segments during development, and it becomes embedded within the nucleus pulposus of the intervertebral disc (IVD) during maturation. The disruption of the notochord band has been observed with IVD degeneration. Since the mechanical competence of the IVD relies on its structural constituents, defining the structure of the notochord during aging is critical for investigations relating to IVD function and homeostasis. The assessment and imaging of the notochord has classically relied on histological techniques, which introduces sectioning artifacts during preparation and spatial biases. Magnetic resonance imaging (MRI) does not offer sufficient resolution to discriminate the notochord from the surrounding the nucleus pulposus, especially in murine models. Current X-ray based computed tomography systems provide imaging resolutions down to the single- and sub- micron scales, and when coupled with contrast-enhancing agents, enable the high-resolution three-dimensional imaging of relatively small features. Utilizing phosphomolybdic acid to preferentially bind to collagen cationic domains, we describe the structure of the notochord remnants with aging in the lumbar IVDs of BALB/c mice. These results provide a highly quantitative and sensitive approach to monitoring the IVD during postnatal development.

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

“…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 metabolized through the mouse quickly, and we observe declines in brae did not exhibit motion artifacts, suggesting that motion was not a factor at these sites.…”

“…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 …”

“…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 contrast‐agents have shown to improve the X‐ray attenuation of IVDs for ex vivo imaging, the nonionic, hydrophilic, and highly biocompatible nature of Ioversol make it particularly suited for in vivo use . Ioversol has been used clinically as a diagnostic radiographic contrast agent.…”

IVD Development: Nucleus Pulposus Development and Sclerotome Specification

“…MRI and X-ray techniques do not offer sufficient resolution to distinguish notochord from developing NP in mice. Using high resolution contrast-enhanced miro-computed tomography (microCT), Bhalla et al have attempted to distinguish these two tissues in mice [29]*. Phosphomolybdic acid (PMA), which targets collagen residues, was used to track changes in the notochord as it developed into the NP.…”

Evaluation of X‐ray tomography contrast agents: A review of production, protocols, and biological applications

“…Ioversol helps to assess the differentiation the relative hydration, whereas PMA reveals the differentiation in collagen composition in disk structures and highlights the end-plates, annulus fibrosus, and the notochord. Another intervertebral investigation was reported byBhalla, Lin, and Tang (2017); the group assessed the postnatal development of murine notochord remnants using micro-CT.Samples were obtained from BALB/c mice of various ages and stained with PMA and micro-CT-scanned. The development and function of the notochord was assessed in different samples for up to 21 days.2.4 | Osmium tetroxideMost favorable molecule-based contrast agents were presented in the previous sections.…”