Human Microstructural Connectomics – Validation with histology and CLARITY
Henriette Rusch1, Maria Morozova2, Katja Reimann1, Alfred Anwander3, Stefan Geyer4, Siawoosh Mohammadi5, Nikolaus Weiskopf6, Thomas Arendt1, Markus Morawski1
1Paul-Flechsig-Institute of Brain Research, University of Leipzig, Leipzig, Germany, 2Max Planck Institute of Human Congnitive and Brain Sciences, Leipzig, Germany, 3Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany, 4Department of Neurophysics, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany, 5University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 6Department of Neurophysics Max Plank Institute for HUman Cognitiveand Brain Sceinces, Leipzig, Germany
The human brain is a network of connecting elements comprising the connectome. It can be differentiated into three spatial scales: the macrostructural level formed by corresponding brain areas and their structural connections via long-range fibers, the mesostructural level formed by e.g. microcircuits and cortical layers and thirdly the microstructural level formed by neurons linked to each other and to glia cells. All spatial levels need to be combined in order to understand the organization of the human brain. To characterize the different spatial scales of the microstructural connectome we need to combine different imaging methods, which are sensitive to different spatial scales and microstructural properties (Morawski et al, 2017). So far, diffusion MRI (dMRI) is the only non-invasive method to characterize macrostructural fiber properties of the brain in vivo. This method alone is limited and can create false results. Therefore, the goal is to verify existing in/ex vivo dMRI of axonal pathways by reconstructing the same fiber tracts en bloc on a microscopic level. To characterize this microstructural cyto- and myeloarchitecture histology is combined with 2D brightfield microscopy as well as CLARITY-lead fluorescence immunohistochemistry with 3D microscopy. The biggest issue is posed by time needed for clearing as the human brain is highly myelinated and pigmented (Braak H, 1979) and the clearing of white matter is a much bigger challenge than grey matter.
A combination of different clearing techniques, immunohistochemistry and 3D microscopy allows for a visualization (330nm – 1µm) of connections over brain samples as thick as 5mm. We obtained human cortex samples at autopsy with prior informed consent (24 hrs post-mortem delay) and approved by the responsible authorities. Brain samples (25 x 25 x 0.5-5 mm) were immersion-fixed with 4% PFA for ~6 weeks. Then tissue blocks underwent an active electrophoreses hydrogel-based clearing procedure first and immunohistochemistry second (CLARITY, Chung et al. 2013) or were bleached, stained and solvent-based cleared afterwards (iDISCO+, Renier et al. 2014; MethBenz). Finally samples were immersed in 47% thiodiethanol for optical index matching. For solvent-based clearing techniques the samples were dehydrated, bleached, stained, dehydrated and cleared with either dichlormethan and dibenzylether or methyl benzoate, depending on the protocol. Antibodies used include HuC/D (neuronal marker), MBP (myelin basic protein marker) and ß-lll-Tubulin (cytoskeleton marker). All samples were imaged either with a Zeiss LSM 880, a LaVision Ultramicroscope II or a 3i Marianas diSPIM microscope. All are equipped with 488nm, 561nm and 640nm laser lines and objectives with magnifications from 2x to 20x, refractive indices from 1.3 to 1.5 and numerical aperture from 0.4 to 1.
All techniques allow for the investigation of the cyto- and myeloarchitecture at subcellular resolution. Focusing on the clearing aspect, the CLARITY method yields the most transparency whereas the iDISCO+ method yields good transparency as well but still shows pigmentation after clearing (Fig.1). 2D microscopy shows parallel arranged cortical fiber tracts in a 30µm thick histological slice (cryo-sectioning). 3D diSPIM microscopy also shows parallel arranged axonal fibers within white and grey matter of a 15 x 16 x 2 mm sized brain tissue block (Fig 2). All samples shown were obtained from the same brain, i.e. taken from the same coronal section of the frontal lobe.
In conclusion, the presented data show that ridding human brain tissue of light scattering lipids is possible via different clearing methods and immunohistochemistry is still applicable beforehand or afterwards. Despite the time and density issue of white matter, transparency can be achieved and 3D images of samples as thick as 5mm show the same results at microscopic resolution as histology. Therefore, a validation of dMRI with the histological ground truth is possible.
Imaging of CLARITY 1
Anatomy and Functional Systems
Cortical Anatomy and Brain Mapping
Cortical Cyto- and Myeloarchitecture 2
White Matter Anatomy, Fiber Pathways and Connectivity
WHITE MATTER IMAGING - DTI, HARDI, DSI, ETC
Other - CLARITY, iDISCO, 3D microscopy
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Chung, K. (2013), 'CLARITY for mapping the nervous system', Nature Methods, vol. 10, no. 6, pp. 508-513
Costantini, I. (2015), 'A versatile clearing agent for multi-modal brain imaging', Scientific Reports, vol. 5, no. 9808, pp. 1-9
Morawski, M. (2017), 'Developing 3D microscopy with CLARITY on human brain tissue: Towards a tool for informing and validating MRI-based histology', NeuroImage, vol. 182, pp. 417-428
Renier, N. (2014), 'iDISCO: A simple, rapid method to immunolabel large tissue samples for volume imaging'. Cell, vol. 159, pp. 896-910