Bringing CLARITY to gray matter atrophy



Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) is a recently developed optical clearing technology that permits intact imaging of the entire brain with minimal protein loss while preserving native fluorescence.

Until now histological studies have focused on a sparse sampling of the tissue in question, as it was not previously possible to do a complete evaluation of these tissues three-dimensionally throughout the entire central nervous system (CNS). With CLARITY, imaging cell populations in 3D in intact tissue is possible.

A common pathological characteristic of axons in both MS and experimental autoimmune encephalomyelitis (EAE) are the presence of ovoids and end bulbs. The purpose of this study was to examine the relationship between gray matter atrophy, as visualized by MRI, and axonal integrity, as visualized by CLARITY. Here, EAE was induced in Thy1-YFP+ mice and in vivo MRI was collected followed by optical clearing of both brains and spinal cords using the “passive clearing” CLARITY technique. EAE was induced in Thy1.1-YFP+ C57BL/6 mice and typical clinical disease was observed, with signs beginning at day 12. In vivo MRIs were collected from five mice with EAE 25 days after disease induction and from six age- and sex-matched healthy controls. The mice were then sacrificed, CLARITY performed on their CNS, and 3D confocal images collected.

The total amount of Thy1-YFP expression was measured and a loss of 20 per cent of Thy1-YFP fluorescence was observed in EAE mice compared to healthy controls. The presence of axonal ovoids is thought to represent disruption of fast axonal transport or alterations of axonal microtubules that lead to Wallerian degeneration. Large numbers of axonal ovoids were observed in the dorsal corticospinal tract of the optically cleared spinal cords in EAE mice. Since CLARITY permits the examination of pathology in intact tissue, the number of axonal ovoids in dorsal corticospinal tract axons over a 5 mm length in three dimensions were quantified.

Sixty one per cent of axons in the dorsal corticospinal tract exhibited ovoids in EAE mice, with virtually none present in normal controls. The number of transected axons that ended in a swollen end bulb were then measured. Eight per cent of axons in the dorsal corticospinal tract terminated in end bulbs in EAE mice, with virtually none in healthy control mice. Intact cerebral hemispheres from mice with EAE and healthy controls were optically cleared using CLARITY and imaged.

The total amount of Thy1-YFP expression in the cerebral cortex was measured and a loss of 17 per cent was observed in EAE mice compared to healthy controls. The total number of layer V Thy1-YFP+ pyramidal neurons in the cerebral cortices were counted. Healthy control mice had a mean of 33,553 ± 274.9 layer V pyramidal neurons, whereas mice with EAE had a mean of 31,157 ± 605.5, a difference of 7%.

Prior to sacrificing the mice, in vivo MRI scans at day 25 of EAE were performed. Volumetry was used to determine if there were any cortical volume differences between EAE and healthy controls. The volume of the cerebral cortex in healthy control mice had a mean of 73.5 ± 0.77 mm3 and mice with EAE had a mean of 68.3 ± 0.99 mm3, a difference of 7 per cent, consistent with previous findings.

A very strong direct correlation between cortical volume and cortical layer V pyramidal neuron number was observed. Further, a very strong negative correlation between in vivo MRI cortical volume and axonal end bulbs in cord was also seen. However, there was no correlation between in vivo MRI cortical volume and axonal ovoids in cord. The use of both MRI and CLARITY provides a unique opportunity to explore the relationship between gray matter atrophy and the pathologies that underlie it. The combination of these techniques allowed the authors to appreciate that there is a relationship between cortical atrophy and axonal end bulbs, but not axonal ovoids, an insight that suggests that there may be a window of reversibility in axonal damage.

Authors: Spence RD, Kurth F
Source: Neuroimage. 2014 Jul 16. pii: S1053-8119(14)00589-8. doi: 10.1016/j.neuroimage.2014.07.017. [Epub ahead of print]
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