Prof. Li-Huei Tsai’s lab at MIT is focused on uncovering the cellular and circuit-level mechanisms that underlie Alzheimer’s disease, the devastating neurological disorder that degrades the brain’s learning and memory systems. In a 2016 paper in Nature (Iaccarino et al.), the lab reported the surprising discovery that by simply entraining the activity of cortical networks at a specific frequency by delivering flashes of light to a mouse’s eyes, the level of amyloid-beta protein, a hallmark of Alzheimer’s neuropathology, could be reduced in the brain’s visual areas.
Additionally, Tsai’s team found that this pattern of activity upregulated expression of genes associated with structural remodeling of microglia, the brain’s immune cells that are thought to play a role in refining synaptic connectivity as well as in removing toxic proteins like amyloid-beta that interfere with circuit function. Interestingly, classical immunohistochemistry experiments carried out on thin sections of brain tissue revealed that driving cortical activity led to increased colocalization of antibody signals identifying both microglia and amyloid-beta plaques, raising the intriguing hypothesis that this network activity could be facilitating microglia’s breakdown of amyloid-beta.
In a paper recently published in Cell (Martorell et al., 2019), the lab explored these mechanisms further by turning to even more cutting-edge histological techniques. In this study, Martorell et al. used brains from a mouse model of Alzheimer’s disease in which amyloid-beta plaques develop in an accelerated fashion. By again delivering patterned sensory stimuli this time in the form of auditory tones, the lab first found using classical immunohistochemistry that this treatment reduced the number and size of plaques in both auditory cortex and the hippocampus, the brain region where memories are first processed. Similarly, they found that auditory stimulation also increased the number and size of microglia in these regions, along with the percentage of microglia showing colocalized amyloid-beta protein signal.
With patterned cortical activity again implicated in facilitating changes associated with microglial removal of amyloid-beta, Tsai’s group next asked whether any other glial cell-types may play a role. Knowing that astrocytes are also involved in waste clean-up, they decided to immunostain for glial fibrillary acidic protein (GFAP) to visualize these cells. To be able to label 100 µm-thick tissue sections with antibodies uniformly and thereby obtain a more unambiguous picture of circuit-level changes stemming from patterned cortical activation, the researchers adopted the chemical engineering-inspired method CLARITY (Chung et al., 2013, Nature) to render fixed brain tissue optically-transparent by removing cell membrane lipids. By using thicker pieces of tissue that capture more of the brain’s structure and connectivity in each slice, Tsai’s team found that the number of GFAP+ astrocytes was decreased in the Alzheimer’s mouse model. Thanks to their use of 100 µm-thick sections versus the 30-40 µm-thick sections used in more traditional approaches, they were able to visualize greater numbers of cells within each region of interest and thereby generate more quantitatively robust data with greater statistical power.
Tsai’s team then thought to pair their new paradigm of patterned auditory stimulation with the visual-based one from their earlier study (Iaccarino et al., 2016, Nature). By delivering synchronized, multisensory stimuli to mice, they found that brain regions beyond each modality’s primary cortical area exhibited entrainment, raising the exciting possibility that increased microglial association with amyloid-beta plaques may result in these areas as well.
When Martorell et al. used immunohistochemistry to evaluate this hypothesis, they made the interesting finding that in the brains of mice that received joint auditory + visual stimulation, a significantly greater number of microglia were found within 25 µm of amyloid-beta plaques as far away as medial prefrontal cortex (mPFC), a brain region important for mediating executive function and other cognitive abilities that are disrupted in Alzheimer’s disease. Notably, plaque number and size were reduced in mPFC as well, suggesting that patterned multisensory stimulation may have a far-reaching and possibly even brain-wide ameliorative effect on plaque load.
To investigate plaque load on the level of a whole mouse brain, Tsai’s group next leveraged two new techniques that together enable remarkably uniform immunostaining of intact organs in less than 2 days. First, they used a novel tissue preservation method called SHIELD (Park et al., 2018, Nature Biotech) that crosslinks biomolecules of interest like amyloid-beta for immunodetection, while allowing cell membrane lipids that scatter light and limit antibody penetration to be removed from the tissue using detergents. SHIELD offers key advantages over CLARITY in that larger tissues can be preserved and cleared with greater consistency and reliability, with SHIELD also providing for superior retention of endogenous fluorescence and the ability to detect a range of biomolecules including nucleic acids.
They applied this advanced technique by SHIELD-preserving Alzheimer’s model brains after the animals had either received joint auditory + visual stimulation for a week or did not (control), and then delipidated the organs in batch using LifeCanvas’ SmartClear II Pro rapid optical-clearing device. By placing the tissue and the detergent solution that bathes it within an electric field, clearing speed is improved dramatically thanks to the field’s ability to facilitate interaction between detergent micelles and the sample’s cell membrane lipids, both of which are charged. As a result, tissues can be cleared in days rather than the 2-3 weeks typical of passive clearing methods. Further, by slowly rotating the sample within the field, each point in the sample gets exposed to the field’s prevailing direction from all 360 degrees, preventing tissue deformation and ensuring that the tissue is cleared evenly. Without rotation, such as in active clearing systems where the electric field is unidirectional and can be too strong, fluorescent proteins can be bleached and the sample can even become burned. LifeCanvas’ SmartClear II Pro active clearing device was designed with the goal of preventing these and other issues that can damage researchers’ precious samples in mind. Interestingly, rotation also has the effect of optimizing the transport of small, freely moving, exogenous materials such as molecular probes deep into the center of the tissue, a feature that is central to the next approach that Tsai’s group used.
Following clearing, Tsai’s lab next applied a new, unpublished technique called eFLASH that combines the principle of stochastic electrotransport (Kim et al., 2015, PNAS) with the SWITCH method (Murray et al., 2015, Cell) for controlling binding kinetics in molecular labeling reactions to immunolabel these intact brains in less than 2 days. By harnessing a rotational electric field and a buffer solution that contains ionic detergents, eFLASH enables antibodies to be driven deep into the tissue actively and under conditions in which binding of the probes to their target sites is initially deterred. Then, by switching the buffer conditions in the middle of the labeling reaction, binding is activated and the sample is labeled from inside out, with labeling density and intensity that are uniform from the sample’s surface to its core.
This new technique was developed by Prof. Kwanghun Chung’s lab at MIT and is compatible with full IgG antibodies and a range of other molecular probes including fluorescent nuclear dyes and lectins. By applying this powerful technique with an amyloid-beta antibody and then imaging the entire brain intact using light-sheet microscopy at single-cell resolution, Iaccarino et al. found using quantitative 3D analysis algorithms (Swaney et al., 2019, bioRxiv) that plaque number and size were dramatically and significantly reduced on a brain-wide basis in animals that had received multisensory stimulation.
By leveraging this pipeline of advanced techniques in which all regions of the sample were processed and analyzed in parallel in one contiguous volume, the researchers were able count every plaque (numbering in the hundreds of thousands per brain!) and make a strong, concrete argument not reliant upon stereological sampling methods that their experimental manipulation led to a robust decrease in plaque load. The algorithm used is open-source (Swaney et al., 2019, bioRxiv) and facilitates alignment of 3D image volumes of mouse cerebral hemispheres and full brains with the Allen Brain Atlas (Lein et al., 2007, Nature), and can be used to quantify labeled plaques, cell bodies, and similar structures and delineate the results by brain region.