Posted on by Dr. Francis Collins
Thousands of Americans are rushed to the hospital each day with traumatic injuries. Daniel Gallego-Perez hopes that small chips similar to the one that he’s touching with a metal stylus in this photo will one day be a part of their recovery process.
The chip, about one square centimeter in size, includes an array of tiny channels with the potential to regenerate damaged tissue in people. Gallego-Perez, a researcher at The Ohio State University Colleges of Medicine and Engineering, Columbus, has received a 2018 NIH Director’s New Innovator Award to develop the chip to reprogram skin and other cells to become other types of tissue needed for healing. The reprogrammed cells then could regenerate and restore injured neural or vascular tissue right where it’s needed.
Gallego-Perez and his Ohio State colleagues wondered if it was possible to engineer a device placed on the skin that’s capable of delivering reprogramming factors directly into cells, eliminating the need for the viral delivery vectors now used in such work. While such a goal might sound futuristic, Gallego-Perez and colleagues offered proof-of-principle last year in Nature Nanotechnology that such a chip can reprogram skin cells in mice. 
Here’s how it works: First, the chip’s channels are loaded with specific reprogramming factors, including DNA or proteins, and then the chip is placed on the skin. A small electrical current zaps the chip’s channels, driving reprogramming factors through cell membranes and into cells. The process, called tissue nanotransfection (TNT), is finished in milliseconds.
To see if the chips could help heal injuries, researchers used them to reprogram skin cells into vascular cells in mice. Not only did the technology regenerate blood vessels and restore blood flow to injured legs, the animals regained use of those limbs within two weeks of treatment.
The researchers then went on to show that they could use the chips to reprogram mouse skin cells into neural tissue. When proteins secreted by those reprogrammed skin cells were injected into mice with brain injuries, it helped them recover.
In the newly funded work, Gallego-Perez wants to take the approach one step further. His team will use the chip to reprogram harder-to-reach tissues within the body, including peripheral nerves and the brain. The hope is that the device will reprogram cells surrounding an injury, even including scar tissue, and “repurpose” them to encourage nerve repair and regeneration. Such an approach may help people who’ve suffered a stroke or traumatic nerve injury.
If all goes well, this TNT method could one day fill an important niche in emergency medicine. Gallego-Perez’s work is also a fine example of just one of the many amazing ideas now being pursued in the emerging field of regenerative medicine.
 Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue. Gallego-Perez D, Pal D, Ghatak S, Malkoc V, Higuita-Castro N, Gnyawali S, Chang L, Liao WC, Shi J, Sinha M, Singh K, Steen E, Sunyecz A, Stewart R, Moore J, Ziebro T, Northcutt RG, Homsy M, Bertani P, Lu W, Roy S, Khanna S, Rink C, Sundaresan VB, Otero JJ, Lee LJ, Sen CK. Nat Nanotechnol. 2017 Oct;12(10):974-979.
Stroke Information (National Institute of Neurological Disorders and Stroke/NIH)
Peripheral Neuropathy (National Institute of Neurological Disorders and Stroke/NIH)
Video: Breakthrough Device Heals Organs with a Single Touch (YouTube)
Gallego-Perez Lab (The Ohio State University College of Medicine, Columbus)
Gallego-Perez Project Information (NIH RePORTER)
NIH Support: Common Fund; National Institute of Neurological Disorders and Stroke
Posted on by Dr. Francis Collins
If laughter really is the best medicine, wouldn’t it be great if we could learn more about what goes on in the brain when we laugh? Neuroscientists recently made some major progress on this front by pinpointing a part of the brain that, when stimulated, never fails to induce smiles and laughter.
In their study conducted in three patients undergoing electrical stimulation brain mapping as part of epilepsy treatment, the NIH-funded team found that stimulation of a specific tract of neural fibers, called the cingulum bundle, triggered laughter, smiles, and a sense of calm. Not only do the findings shed new light on the biology of laughter, researchers hope they may also lead to new strategies for treating a range of conditions, including anxiety, depression, and chronic pain.
In people with epilepsy whose seizures are poorly controlled with medication, surgery to remove seizure-inducing brain tissue sometimes helps. People awaiting such surgeries must first undergo a procedure known as intracranial electroencephalography (iEEG). This involves temporarily placing 10 to 20 arrays of tiny electrodes in the brain for up to several weeks, in order to pinpoint the source of a patient’s seizures in the brain. With the patient’s permission, those electrodes can also enable physician-researchers to stimulate various regions of the patient’s brain to map their functions and make potentially new and unexpected discoveries.
In the new study, published in The Journal of Clinical Investigation, Jon T. Willie, Kelly Bijanki, and their colleagues at Emory University School of Medicine, Atlanta, looked at a 23-year-old undergoing iEEG for 8 weeks in preparation for surgery to treat her uncontrolled epilepsy . One of the electrodes implanted in her brain was located within the cingulum bundle and, when that area was stimulated for research purposes, the woman experienced an uncontrollable urge to laugh. Not only was the woman given to smiles and giggles, she also reported feeling relaxed and calm.
As a further and more objective test of her mood, the researchers asked the woman to interpret the expression of faces on a computer screen as happy, sad, or neutral. Electrical stimulation to the cingulum bundle led her to see those faces as happier, a sign of a generally more positive mood. A full evaluation of her mental state also showed she was fully aware and alert.
To confirm the findings, the researchers looked to two other patients, a 40-year-old man and a 28-year-old woman, both undergoing iEEG in the course of epilepsy treatment. In those two volunteers, stimulation of the cingulum bundle also triggered laughter and reduced anxiety with otherwise normal cognition.
Willie notes that the cingulum bundle links many brain areas together. He likens it to a super highway with lots of on and off ramps. He suspects the spot they’ve uncovered lies at a key intersection, providing access to various brain networks regulating mood, emotion, and social interaction.
Previous research has shown that stimulation of other parts of the brain can also prompt patients to laugh. However, what makes stimulation of the cingulum bundle a particularly promising approach is that it not only triggers laughter, but also reduces anxiety.
The new findings suggest that stimulation of the cingulum bundle may be useful for calming patients’ anxieties during neurosurgeries in which they must remain awake. In fact, Willie’s team did so during their 23-year-old woman’s subsequent epilepsy surgery. Each time she became distressed, the stimulation provided immediate relief. Also, if traditional deep brain stimulation or less invasive means of brain stimulation can be developed and found to be safe for long-term use, they may offer new ways to treat depression, anxiety disorders, and/or chronic pain.
Meanwhile, Willie’s team is hard at work using similar approaches to map brain areas involved in other aspects of mood, including fear, sadness, and anxiety. Together with the multidisciplinary work being mounted by the NIH-led BRAIN Initiative, these kinds of studies promise to reveal functionalities of the human brain that have previously been out of reach, with profound consequences for neuroscience and human medicine.
 Cingulum stimulation enhances positive affect and anxiolysis to facilitate awake craniotomy. Bijanki KR, Manns JR, Inman CS, Choi KS, Harati S, Pedersen NP, Drane DL, Waters AC, Fasano RE, Mayberg HS, Willie JT. J Clin Invest. 2018 Dec 27.
Video: Patient’s Response (Bijanki et al. The Journal of Clinical Investigation)
Epilepsy Information Page (National Institute of Neurological Disease and Stroke/NIH)
Jon T. Willie (Emory University, Atlanta, GA)
NIH Support: National Institute of Neurological Disease and Stroke; National Center for Advancing Translational Sciences
Posted on by Dr. Francis Collins
Credit: Gao et. al, Science
Researchers are making amazing progress in developing new imaging approaches. And they are now using one of their latest creations, called ExLLSM, to provide us with jaw-dropping views of a wide range of biological systems, including the incredibly complex neural networks within the mammalian brain.
In this video, ExLLSM takes us on a super-resolution, 3D voyage through a tiny sample (0.0030 inches thick) from the part of the mouse brain that processes sensation, the primary somatosensory cortex. The video zooms in and out of densely packed pyramidal neurons (large yellow cell bodies), each of which has about 7,000 synapses, or connections. You can also see presynapses (cyan), the part of the neuron that sends chemical signals; and postsynapes (magenta), the part of the neuron that receives chemical signals.
At 1:45, the video zooms in on dendritic spines, which are mushroom-like nubs on the neuronal branches (yellow). These structures, located on the tips of dendrites, receive incoming signals that are turned into electrical impulses. While dendritic spines have been imaged in black and white with electron microscopy, they’ve never been presented before on such a vast, colorful scale.
The video comes from a paper, published recently in the journal Science , from the labs of Ed Boyden, Massachusetts Institute of Technology, Cambridge, and the Nobel Prize-winning Eric Betzig, Janelia Research Campus of the Howard Hughes Medical Institute, Ashburn, VA. Like many collaborations, this one comes with a little story.
Four years ago, the Boyden lab developed expansion microscopy (ExM). The technique involves infusing cells with a hydrogel, made from a chemical used in disposable diapers. The hydrogel expands molecules within the cell away from each other, usually by about 4.5 times, but still locks them into place for remarkable imaging clarity. It makes structures visible by light microscopy that are normally below the resolution limit.
Though the expansion technique has worked well with a small number of cells under a standard light microscope, it hasn’t been as successful—until now—at imaging thicker tissue samples. That’s because thicker tissue is harder to illuminate, and flooding the specimen with light often bleaches out the fluorescent markers that scientists use to label proteins. The signal just fades away.
For Boyden, that was a problem that needed to be solved. Because his lab’s goal is to trace the inner workings of the brain in unprecedented detail, Boyden wants to image entire neural circuits in relatively thick swaths of tissue, not just look at individual cells in isolation.
After some discussion, Boyden’s team concluded that the best solution might be to swap out the light source for the standard microscope with a relatively new imaging tool developed in the Betzig lab. It’s called lattice light-sheet microscopy (LLSM), and the tool generates extremely thin sheets of light that illuminate tissue only in a very tightly defined plane, dramatically reducing light-related bleaching of fluorescent markers in the tissue sample. This allows LLSM to extend its range of image acquisition and quickly deliver stunningly vivid pictures.
Telephone calls were made, and the Betzig lab soon welcomed Ruixuan Gao, Shoh Asano, and colleagues from the Boyden lab to try their hand at combining the two techniques. As the video above shows, ExLLSM has proved to be a perfect technological match. In addition to the movie above, the team has used ExLLSM to provide unprecedented views of a range of samples—from human kidney to neuron bundles in the brain of the fruit fly.
Not only is ExLLSM super-resolution, it’s also super-fast. In fact, the team imaged the entire fruit fly brain in 2 1/2 days—an effort that would take years using an electron microscope.
ExLLSM will likely never supplant the power of electron microscopy or standard fluorescent light microscopy. Still, this new combo imaging approach shows much promise as a complementary tool for biological exploration. The more innovative imaging approaches that researchers have in their toolbox, the better for our ongoing efforts to unlock the mysteries of the brain and other complex biological systems. And yes, those systems are all complex. This is life we’re talking about!
 Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution. Gao R, Asano SM, Upadhyayula S, Pisarev I, Milkie DE, Liu TL, Singh V, Graves A, Huynh GH, Zhao Y, Bogovic J, Colonell J, Ott CM, Zugates C, Tappan S, Rodriguez A, Mosaliganti KR, Sheu SH, Pasolli HA, Pang S, Xu CS, Megason SG, Hess H, Lippincott-Schwartz J, Hantman A, Rubin GM, Kirchhausen T, Saalfeld S, Aso Y, Boyden ES, Betzig E. Science. 2019 Jan 18;363(6424).
Video: Expansion Microscopy Explained (YouTube)
Video: Lattice Light-Sheet Microscopy (YouTube)
How to Rapidly Image Entire Brains at Nanoscale Resolution, Howard Hughes Medical Institute, January 17, 2019.
Synthetic Neurobiology Group (Massachusetts Institute of Technology, Cambridge)
Eric Betzig (Janelia Reseach Campus, Ashburn, VA)
NIH Support: National Institute of Neurological Disorders and Stroke; National Human Genome Research Institute; National Institute on Drug Abuse; National Institute of Mental Health; National Institute of Biomedical Imaging and Bioengineering