It’s not Harry Potter magic. But all the same, in the July 31 Cell, researchers describe a technique that allows whole cellular networks to come into view in situ. The technique turns entire rodents see-through and permeable, granting open optical access to every nook, cranny, and neuron in the body. Called “whole-body clearing,” the method strips away the lipids and membranes that normally limit the depths to which light from microscopes can probe. With this technique, researchers can trace neural networks within the brain outward to those that extend to organs throughout the body, and avoid the painstaking procedure of imaging thin slices of tissue one at a time. The whole-body technique one-ups previous studies in which researchers made an entire rodent brain transparent.
“There is a very large effort to understand the brain connectome; however, there is very little known about the peripheral nervous system,” senior author Viviana Gradinaru of the California Institute of Technology in Pasadena told Alzforum. Understanding the diverse neural networks that control vital organs will provide a better foundation for learning how best to treat all manner of diseases, she added. For that to happen, researchers need the ability to image vast stretches of networks that run from the brain to organs and back, a task too daunting for researchers to perform slice by slice.
The concept of tissue clearing, which involves destroying lipid membranes with detergents to render the tissue transparent, is nothing new. Alas, older techniques often damaged tissue or altered its morphology. More recent techniques have managed to keep tissues intact, but the chemicals used for clearing made the tissue impermeable to staining reagents such as fluorescently tagged antibodies. Limitations in the working depth of microscopes also stymied motivation to create organ- or body-clearing methods amenable to imaging, according to Gradinaru. “With recent advances in microscopy and imaging, I would say the time is prime for this technique now,” she said.
As a staff scientist at Stanford University, Gradinaru helped develop the CLARITY method, which made a rodent brain transparent and permeable enough to allow access to antibodies for staining(see related April 2013 news story on the Alzforum). In that study, researchers embedded the brain in a polymerizing gel, then used an electrical current to promote the distribution of detergents throughout the tissue, rendering it transparent. The problem with that method, Gradinaru said, was that if the conditions were not “just right,” the current could burn the tissue.
Wanting to create both a gentler clearing method and render an entire body transparent, first author Bin Yang and colleagues started off by tweaking their gel and detergent solutions and testing them on tissue slices. The researchers settled on a reagent combination that created a gel with pores large enough for detergent to pass through without the added electrical jolt. Using this “Goldilocks” protocol, which the researchers dubbed PACT (passive CLARITY), they stained and imaged 1- to 3-mm thick slices of brain, kidney, heart, lung, and intestine. Besides various tissue-specific antibodies and dyes, the researchers also labeled mRNA in cleared brain slices with fluorescent oligonucleotide probes. Uncleared brain slices had high background noise due to tissue auto-fluorescence; in contrast, the transparent slices enabled visualization of single molecules of β-actin mRNA. Gradinaru hopes the technique will one day facilitate the analysis of global gene expression throughout organs.
To scale up the PACT method to the entire rodent body, the researchers exploited the vasculature to deliver hydrogel and clearing solutions. Using a pump connected to a cardiac catheter, the researchers cycled clearing solutions throughout mice and rats for one or two weeks, respectively, until the rodents’ entire bodies became transparent. The researchers then delivered fluorescent antibodies and dyes via the same perfusion system to the whole body for imaging. The researchers called this method PARS (perfusion-assisted agent release in situ). Using it in mice expressing enhanced green fluorescent protein (eGFP) under the Thy1 promoter, the researchers were able to peer through standard confocal microscopes deep into the centers of multiple organs—including brain, spinal cord, kidney, lung, liver, and pancreas—and discern subcellular detail.
The blood-brain barrier remained a formidable foe. Despite clearing, it still prevented efficient delivery of full-size antibodies to the rodent brain, Gradinaru said. However, when the researchers used PARS to deliver GFAP-specific nanobodies—single-domain antibody fragments about one-tenth the size of full antibodies—glial cells in the brain vasculature and in surrounding tissue were extensively labeled. Unfortunately, nanobodies are not yet available for the full complement of brain proteins, Gradinaru said. Once they are, nanobodies will greatly expand the utility of the PARS technique to access the brain along with other organs in the body.
To get around this limitation now, the researchers developed a brain-specific PARS technique—PARS-CSF—in which they cycled clearing fluids directly into the cerebrospinal fluid in different areas of interest throughout the central nervous system. That way, the researchers cleared the brain or spinal cord in four days. PARS-CSF could be useful for researchers who want to label different regions of the brain or spinal cord without clearing the entire body, Gradinaru said.
Gradinaru plans to use the new techniques to trace degeneration of cells throughout the brain and body of rodent models of disease. Tracking which cells harbor pathogenic proteins or show signs of death throughout the course of a disease such as Parkinson’s, Gradinaru said, could prove useful in understanding how and where the disease originates and spreads. Some hypothesize that Parkinson’s disease originates in the gut and spreads to the brain via the vagus nerve, a complex nerve with myriad fibers. The vagus nerve also has been implicated in depression, Gradinaru said, so taking a whole-body approach would be particularly useful in understanding how this complex nerve mediates disorders. She said the ultimate goal is to understand such networks well enough to target them with electrical stimulation in just the right spots to treat disease.
Image: Visualization of YFP-labeled neurons in 1 mm PACT cleared mouse spinal cord slice. Credit: Yang et al., 2014.
Cell. 2014 Aug 14; 158(4):945-58. Epub 2014 Jul 31.
Viviana Gradinaru’s laboratory has published a fascinating article describing a method by which organs or whole bodies can be rendered transparent and made permeable for immunohistochemistry, FISH, and high-resolution microscopy. This enables researchers to study the molecular composition of organs and tissues, and at the same time, their connectivity. For example, antibodies could penetrate through most of a 1 mm mouse brain section, giving brilliant immunofluorescence with a triple stain, visualizing blood vessels, astrocytes, and microglia, and showing their relationship in an intact slice of brain tissue. Or, brain and spinal neurons can be visualized in thick sections, clearly showing their connectivity and dendritic spines. While the authors are speculating about the use of these novel techniques in cancer research and neurodegenerative diseases, there are a number of questions in pain research that could be answered with the help of these techniques. For example, the dispute about sprouting Aβfibers connecting to spinal nociceptors as a cause of allodynia might have been solved more quickly with high-resolution immunofluorescence in thick spinal cord sections.
In pain research, we have become quite good at finding out where things happen (e.g., with functional MRI studies) and, on the other hand, at analyzing what happens (e.g., with gene expression and proteomic studies). The challenge is to bring the information together. Ideally, fMRI would allow us to identify active brain regions upon a painful stimulus; multiple single unit recording would give us information on the activity of individual neurons; and high-resolution morphology would afterwards allow us to identify the anatomical and neurochemical connections.
Another nice application would be the nociceptive innervation in the periphery. While we have learned a lot about nociceptive skin innervation, the currently available morphological techniques still do not allow us to see the connections from nerve fiber bundles to the thin nociceptive endings, and we still cannot unequivocally identify a nociceptor just by its morphological appearance. Here, a better view of the fiber’s connections, i.e., up to the spinal cord, would answer many questions.
Muscle nociceptors are not well described morphologically, most likely due to technical issues, given that they are located in the internal and external perimysium. Transparent thick sections with high-resolution immunofluorescence might elucidate the morphology and distribution of these nociceptors.
In the spinal cord, the question of inhibitory or excitatory control, for example, by serotonergic neurons, might get renewed input by better anatomical resolution of descending tracts and their segmental targets, combined with information on the neurotransmitters present in the terminals.
Thus, we can hope that pain researchers will take up Dr. Gradinaru’s methods and put them to good use to map brain-body connections relevant to pain.