Brain virtual histology with X-ray phase-contrast tomography Part I: whole-brain myelin mapping in white-matter injury models

Abstract

White-matter injury leads to severe functional loss in many neurological diseases. Myelin staining on histological samples is the most common technique to investigate white-matter fibers. However, tissue processing and sectioning may affect the reliability of 3D volumetric assessments. The purpose of this study was to propose an approach that enables myelin fibers to be mapped in the whole rodent brain with microscopic resolution and without the need for strenuous staining. With this aim, we coupled in-line (propagation-based) X-ray phase-contrast tomography (XPCT) to ethanol-induced brain sample dehydration. We here provide the proof-of-concept that this approach enhances myelinated axons in rodent and human brain tissue. In addition, we demonstrated that white-matter injuries could be detected and quantified with this approach, using three animal models: ischemic stroke, premature birth and multiple sclerosis. Furthermore, in analogy to diffusion tensor imaging (DTI), we retrieved fiber directions and DTI-like diffusion metrics from our XPCT data to quantitatively characterize white-matter microstructure. Finally, we showed that this non-destructive approach was compatible with subsequent complementary brain sample analysis by conventional histology. In-line XPCT might thus become a novel gold-standard for investigating white-matter injury in the intact brain. This is Part I of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part II shows how in-line XPCT enables the whole-brain 3D morphometric analysis of amyloid- β (A β ) plaques.

Why are amyloid-β plaques detected by X-ray phase-contrast imaging? Role of metals revealed through combined synchrotron infrared and X-ray fluorescence microscopies

“…Ring artifacts were removed using an in-house tool implemented by the company NOVITOM (France; https://www.novitom.com/en/) [19]. Samples were isolated from the background (air, glass tube and ethanol) and rotated within the AMIRA 3D software (release 2021.1, Thermo Fisher Scientific, USA).…”

“…Complete acquisition parameters are reported in Supplemental Table 1. Human samples were imaged at the ESRF, beamline ID-19, using similar technique and parameters, which were previously reported [19]. 7…”

“…X-ray 3D imaging of Aβ plaques has been described in transgenic rodent models that develop amyloidosis for the past decade, using propagation-based phase contrast (either with synchrotron radiation [5, 6, 7, 8, 9, 10] or with a laboratory source [11]) or using gratingbased phase contrast (with synchrotron radiation [12, 13, 14, 5]). These techniques generate 3D images with an isotropic resolution ranging from 1 µm to 10 µm, thus providing a so-called “virtual histology” of the excised brain [6, 15, 16, 17, 18, 19] with minimal preparation (fixation, dehydration or paraffin embedding). Imaging AD samples with XPCT is particularly interesting because plaques stand out in the images without the need to add any staining agents.…”

Revealing the three-dimensional murine brain microstructure by contrast-enhanced computed tomography

“…A diffusion-tensor imaging (DTI)-like algorithm was applied, developed by NOVITOM. 1 The algorithm, previously described by 1 https://www.novitom.com/en/ Chourrout et al (2022a), was developed based on image gradient analysis to detect the orientation of white-matter fibers. We selected the following input parameters, adapted to the mouse brain: gradient orientation (0.5), gradient orientation tensor (0.5) and fiber orientation tensor (5).…”

“…However, to obtain a high discriminative power, methods for enhancing the contrast between different tissue constituents are required. At present, the most promising contrast-enhancing methods include phase-contrast microCT (PCCT) ( Takeda et al, 2013 ; Barbone et al, 2021 ; Rodgers et al, 2021 , 2022 ; Chourrout et al, 2022a , b ; Palermo et al, 2022 ) and contrast-enhanced microCT (CECT) ( Rodrigues et al, 2021 ). While PCCT makes use of the refractive properties of the X-rays, CECT aims to increase the X-ray attenuating properties of different tissue constituents (e.g., white matter versus gray matter) by incubating or infiltrating the tissue with contrast-enhancing staining agents (CESAs).…”