Now a team led by researchers at the Georgia Institute of Technology has engineered a way of studying the barrier more closely with the intent of helping drug developers do the same. In a new study, the researchers cultured the human blood-brain barrier on a chip, recreating its physiology more realistically than predecessor chips.
The new chip devised a healthy environment for the barrier’s central component, a brain cell called the astrocyte, which is not a neuron, but which acts as neurons’ intercessors with the circulatory system. Astrocytes interface in human brains with cells in the vasculature called endothelial cells to collaborate with them as the blood-brain barrier.
But astrocytes are a particularly fussy partner, which makes them a great part of the gatekeeper system but also challenging to culture in a physiologically accurate manner. The new chip catered to astrocytes’ sensibilities by culturing in 3D instead of in a flat manner, or 2D.
The 3D space allowed astrocytes to act more naturally, and this improved the whole barrier model by also allowing cultured endothelial cells to function better. The new chip presented researchers with more healthy blood-brain barrier functions to observe than in previous barrier models.
“You need to be able to closely mimic a tissue on a chip in a healthy status and in homeostasis. If we can’t model the healthy state, we can’t really model disease either, because we have no accurate control to measure it against,” said YongTae Kim, an associate professor in Georgia Tech’s George W. Woodruff School of Mechanical Engineering and the study’s principal investigator.
In the new chip, the astrocytes even looked more natural in the 3D space, unfolding the star-like shape that gives them their “astro” name. In the 2D cultures, by contrast, astrocytes looked like fried eggs with fringes. With this 3D setting, the chip has added possibilities for reliable research of the human blood-brain barrier, where currently alternatives are few.
“No animal model comes close enough to the intricate function of the human blood-brain barrier. And we need better human models because experimental drugs that have successfully entered animal brains have failed at the human barrier,” Kim said.
The team published its results on January 10, 2020, in the journal Nature Communications. The research was funded by the National Institutes of Health. Kim has founded a company with plans to mass-produce the new chip in the future for use in academic and potentially pharmaceutical research.
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The brain is the only part of the body outfitted with astrocytes, which regulate nourishment uptake and waste removal in their own, unique way.
“Upon the brain’s request, astrocytes collaborate with the vasculature in real-time what the brain needs and opens its gates to let in only that bit of water and nutrients. Astrocytes go to get just what the brain needs and don’t let much else in,” Kim said.
Astrocytes form a protein structure called aquaporin-4 in their membranes that are in contact with vasculature to let in and out water molecules, which also contributes to clearing waste from the brain.
“In previous chips, aquaporin-4 expression was not observed. This chip was the first,” Kim said. “This could be important in researching Alzheimer’s disease because aquaporin-4 is important to clearing broken-down junk protein out of the brain.”
One of the study’s co-authors, Dr. Allan Levey from Emory University, a highly cited researcher in neurological medicine, is interested in the chip’s potential in tackling Alzheimer’s. Another, Dr. Tobey McDonald, also of Emory, researches pediatric brain cancer and is interested in the chip’s possibilities in studying the delivery of potential brain cancer treatments.
Astrocytes also gave signs that they were healthier in the chip’s 3D cultures than in 2D cultures by expressing less of a gene triggered by pathology.
“Astrocytes in 2D culture expressed significantly higher levels of LCN2 than those in 3D. When we cultured in 3D, it was only about one fourth as much,” Kim said.
The healthier state also made astrocytes better able to show an immune reaction.
“When we purposely confronted the astrocyte with pathological stress in a 3D culture, we got a clearer reaction. In 2D, the ground state was already less healthy, and then the reaction to pathological stresses did not come across so clearly. This difference could make the 3D culture very interesting for pathology studies.”
In testing related to drug delivery, nanoparticles moved through the blood-brain-barrier after engaging endothelial cell receptors, which caused these cells to engulf the particles then transport them to what would be inside the human brain in a natural setting. This is part of how endothelial cells worked better when connected to astrocytes cultured in 3D.
“When we inhibited the receptor, the majority of nanoparticles wouldn’t make it in. That kind of test would not work in animal models because of cross-species inaccuracies between animals and humans,” Kim said. “This was an example of how this new chip can let you study the human blood-brain barrier for potential drug delivery the way you can’t in animal models.”
Also Read: Flickering Light Mobilizes Brain Chemistry That May Fight Alzheimer’s
These researchers also coauthored the study: Song Ih Ahn, Yoshitaka Sei, Hyun-Ji Park, Jinhwan Kim, Yujung Ryu, and Jeongmoon Choi, and Hak-Joon Sung of Georgia Tech. The research was funded by National Institutes of Health’s Director’s New Innovator Award (1DP2HL142050), the National Institute of Neurological Disorders and Stroke (grant R21NS091682), and the National Institutes on Aging (grant R21AG056781). Tony Kim is also affiliated with Georgia Tech’s Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory, Georgia Tech’s Parker H. Petit Institute for Bioengineering and Bioscience, and Georgia Tech’s Institute for Electronics and Nanotechnology. Any findings, conclusions, and recommendations are those of the authors and not necessarily of the National Institutes of Health.
Writer & Media Representative: Ben Brumfield (404-272-2780), email: ben.brumfield@comm.gatech.edu
Georgia Institute of Technology
]]>Now, researchers have tapped into how the flicker may work. They discovered in the lab that the exposure to light pulsing at 40 hertz – 40 beats per second – causes brains to release a surge of signaling chemicals that may help fight the disease.
Though conducted on healthy mice, this new study is directly connected to human trials, in which Alzheimer’s patients are exposed to 40 Hz light and sound. Insights gained in mice at the Georgia Institute of Technology are informing the human trials in collaboration with Emory University.
“I’ll be running samples from mice in the lab, and around the same time, a colleague will be doing a strikingly similar analysis on patient fluid samples,” said Kristie Garza, the study’s first author. Garza is a graduate research assistant in the lab of Annabelle Singer at Georgia Tech and also a member of Emory’s neuroscience program.
One of the surging signaling molecules in the new study on mice is strongly associated with the activation of brain immune cells called microglia, which purge an Alzheimer’s hallmark – amyloid beta plaque, junk protein that accumulates between brain cells.
In 2016, researchers discovered that light flickering at 40 Hz mobilized microglia in mice afflicted with Alzheimer’s to clean up that junk. The new study looked for brain chemistry that connects the flicker with microglial and other immune activation in mice and exposed a surge of 20 cytokines – small proteins secreted externally by cells and which signal to other cells. Accompanying the cytokine release, internal cell chemistry – the activation of proteins by phosphate groups – left behind a strong calling card.
“The phosphoproteins showed up first. It looked as though they were leading, and our hypothesis is that they triggered the release of the cytokines,” said Singer, who co-led the new study and is an assistant professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory.
“Beyond cytokines that may be signaling to microglia, a number of factors that we identified have the potential to support neural health,” said Levi Wood, who co-led the study with Singer and is an assistant professor in Georgia Tech’s George W. Woodruff School of Mechanical Engineering.
The team published its findings in the Journal of Neuroscience on February 5, 2020. The research was funded by the National Institute of Neurological Disorders and Stroke at the National Institutes of Health, and by the Packard Foundation.
Singer was co-first author on the original 2016 study at the Massachusetts Institute of Technology, in which the therapeutic effects of 40 Hz were first discovered in mice.
Alzheimer’s strikes, with few exceptions, late in life. It destroys up to 30% of a brain’s mass, carving out ravines and depositing piles of amyloid plaque, which builds up outside of neurons. Inside neurons, phosphorylated tau protein forms similar junk known as neurofibrillary tangles suspected of destroying mental functions and neurons.
After many decades of failed Alzheimer’s drug trials costing billions, flickering light as a potentially successful Alzheimer’s therapy seems surreal even to the researchers.
“Sometimes it does feel like science fiction,” Singer said.
The 40 Hz frequency stems from the observation that brains of Alzheimer’s patients suffer early on from a lack of what is called gamma, moments of gentle, constant brain waves acting like a dance beat for neuron activity. Its most common frequency is right around 40 Hz, and exposing mice to light flickering at that frequency restored gamma and also appears to have prevented heavy Alzheimer’s brain damage.
Adding to the surrealness, gamma has also been associated with esoteric mind expansion practices, in which practitioners perform light and sound meditation. Then, in 2016, research connected gamma to working memory, a function key to train of thought.
In the current study, the surging cytokines hinted at a connection with microglial activity, and in particular, the cytokine Macrophage Colony-Stimulating Factor (M-CSF).
“M-CSF was the thing that yelled, ‘Microglia activation!’” Singer said.
The researchers will look for a causal connection to microglia activation in an upcoming study, but the overall surge of cytokines was a good sign in general, they said.
“The vast majority of cytokines went up, some anti-inflammatory and some inflammatory, and it was a transient response,” Wood said. “Often, a transient inflammatory response can promote pathogen clearance; it can promote repair.”
“Generally, you think of an inflammatory response as being bad if it’s chronic, and this was rapid and then dropped off, so we think that was probably beneficial,” Singer added.
The 40 Hz stimulation did not need long to trigger the cytokine surge.
“We found an increase in cytokines after an hour of stimulation,” Garza said. “We saw phosphoprotein signals after about 15 minutes of flickering.”
Perhaps about 15 minutes was enough to start processes inside of cells and about 45 more minutes were needed for the cells to secrete cytokines. It is too early to know.
As controls, the researchers applied three additional light stimuli, and to their astonishment, all three had some effect on cytokines. But stimulating with 20 Hz stole the show.
“At 20 Hz, cytokine levels were way down. That could be useful, too. There may be circumstances where you want to suppress cytokines,” Singer said. “We’re thinking different kinds of stimulation could potentially become a platform of tools in a variety of contexts like Parkinson’s or schizophrenia. Many neurological disorders are associated with immune response.”
The research team warns against people improvising light therapies on their own, since more data is needed to thoroughly establish effects on humans, and getting frequencies wrong could possibly even do damage.
Also read: A family coping with Alzheimer’s leads you through our fight against it
Also read: Why Alzheimer’s research probably needs to shift focus
Lu Zhang and Ben Borron from the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University co-authored the study. The research was funded by the National Institute of Neurological Disorders and Stroke at the National Institutes of Health (grants NIH R01-NS109226 and R01-NS109226-01S1), by the Packard Foundation, the Friends and Alumni of Georgia Tech, and by the Lane family. Any findings, conclusions, and recommendations are those of the authors and not necessarily of the sponsors.
Writer & Media Representative: Ben Brumfield (404-272-2780), email: ben.brumfield@comm.gatech.edu
Georgia Institute of Technology
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Cerebral palsy (CP) is the most common cause of physical disability in children in most developed countries, and spastic CP is the most common form of the disorder. For these patients (and others), spasticity can be severely debilitating, negatively impacting their movement, speech, gait, and overall quality of life.
The lab of Lena Ting, professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory, and in the Division of Physical Therapy in Emory’s Department of Rehabilitation Medicine, is tackling the problem, shedding new light on issues underlying spasticity.
Ting’s lab is part of an international collaborative effort with a recently published research article in the open access scientific journal, PLOS One. She is corresponding author of, “Interaction between muscle tone, short-range stiffness and increased sensory feedback
gains explains key kinematic features of the pendulum test in spastic cerebral palsy: A
simulation study.”
The pendulum test is a sensitive clinical assessment of spasticity in which the lower leg is
dropped from the horizontal position and the features of leg motion are recorded. “This problem actually arose out of a homework problem for my Computational Neuromechanics class, where we simulate the leg as a pendulum,” said Ting.
In typically-developed people, the swinging leg behaves like a damped pendulum, with the angle of leg swing decreasing as it oscillates several times before coming to rest. In children with spastic CP, three key differences in the leg motion are observed: Reduced angle of leg swing in the first oscillation, fewer oscillations, and the coming to rest at a less vertical angle.
Overall, the decrease in the first swing has been found to be the best predictor of spasticity severity, but why this is the case is has not been clear. Ting’s team hypothesized that increased muscle tone– the continual contraction of muscles while at rest–accounts for both the reduced leg swing and the non-vertical resting leg angle. This idea contrasts with the clinical explanation of spasticity as an abnormal increase in the activation of reflexes as the leg is stretched with higher velocities.
“We were stumped because the clinical explanation of increased velocity-dependent reflexes didn’t generate realistic motion,” Ting said. “But we happened to be working on a different research project studying an interesting property of muscles called short-range stiffness, which increases when muscles are activated. We wanted to know if this very rapid rise and drop of resistive force in muscles when they are stretched could explain the parts of the pendulum test that were giving us a hard time in the simulation.”
So the researchers developed and tested a physiologically-plausible computer simulation of how muscle tone and reflexes would interact to reproduce key features of the pendulum test for spasticity across a range of severity levels. Their new model helps to explain a whole range of pendulum test kinematics in people with and without CP.
“Increased muscle tone plays a primary role in generating a key feature of the leg motion that is most closely related to the level of spasticity,” Ting explained. “Even when reflexes are increased, can only account for pendulum test results across the spectrum of spasticity severity if we also increase muscle tone and short-range stiffness. This is exciting because the pendulum test is more objective than a clinician’s subjective assessment of leg stiffness. And with our model we can now begin to understand how multiple mechanisms of spasticity might interact to cause abnormal body motion, not just in the pendulum test, but in everyday movements.”
Lead author of the paper was Friedl De Groote, assistant professor in the Department of Movement Sciences at KU Leuven in Belgium. Other authors were both researchers from Ting’s lab, Kyle Blum and Brian Horslen.
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“This year’s awardees reflect the cutting–edge research being done at every career stage across neurology and neuroscience,” said David M. Holtzman, MD, president of the ANA and Andrew B. and Gretchen P. Jones Professor and Chairman, Dept. of Neurology at the Washington University School of Medicine. “The pace of advances we’re seeing in translational neuroscience and the neurobiology of disease are extraordinary. We hope the gains will inspire the new generation of physician scientists to pursue careers that combine research and teaching with clinical practice. There has never been a more exciting time to work in academic neurology.”
Each year, the ANA Annual Meeting convenes more than 900 of the nation's top academic neurologists and neuroscientists to share updates and late-breaking research on the diseases that affect more than 100 million Americans each year including stroke, Alzheimer’s disease, Parkinson’s disease, neuromuscular disorders, headache, traumatic brain and spinal cord injuries, epilepsy, multiple sclerosis and more.
Derek Denny-Brown Young Neurological Scholars
The Derek Denny-Brown Young Neurological Awards are clinical awards given each year during the Annual Meeting to new members of the association who have achieved significant stature in neurological research, and who show promise and will continue making major contributions to the field of neurology.
The Derek Denny-Brown Young Neurological Scholar Award in Neuroscience went to Cassie S. Mitchell, Ph.D., Georgia Institute of Technology. Her presentation title: Literature-based discovery facilitates predictive medicine for neurological disease.
Full list of ANA 2019 winners.
]]>But a team of researchers at the Georgia Institute of Technology wants develop intelligent closed-loop algorithms for turning measurements into precise actions in real time – kind of like those used in technologies such as self-driving cars and robotics.
"Intelligent algorithms for interacting with the brain holds promise to help us alleviate diseases with no current treatments, as well as better understanding the basis of human intelligence,” says Chris Rozell, professor in Georgia Tech’s School of Electrical and Computer Engineering, who is leading the study with Garrett Stanley, professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University. Both are researchers in the the Petit Institute for Bioengineering and Bioscience at Georgia Tech, where Rozell also is a member of the Center for Machine Learning and Stanley is co-director of the Neural Engineering Center.
Their project just received a $1.6 million, five-year award from the National Institutes of Health (NIH)/National Institutes of Neurological Disorders and stroke (NINDS) through a long-standing and innovative NSF/NIH partnership in the Collaborative Research in Computational Neuroscience (CRCNS) program. The project leverages neurotechnology and engineering advances to pioneer a nascent field, closed-loop computational neuroscience.
"There has been an explosion of remarkable advances in both neuroengineering and machine learning, making the intersection of these fields one of the most exciting frontiers I can imagine,” adds Rozell. “I am excited that this collaborative project will allow us to pioneer these interactive neurotechnology advances.”
Rather than just analyzing data after an experiment, the team’s integrative approach will develop real-time algorithms that operate as a type of autopilot for a neural circuit, “where we can lock in a precise response, regardless of surrounding activity,” Rozell says.
The five-year project, called “Closed-Loop Computational Neuroscience for Causally Dissecting Circuits,” will build on the theory, methods, and findings of engineering, computer science, neuroscience, and other disciplines (machine learning and genetics, for example). Through the CRCNS program, the National Science Foundation and National Institutes of Health (along with several international partners) support collaborative activities designed to advance understanding of nervous system structure and function, the mechanisms underlying nervous system disorders, and the computational strategies used by the nervous system.
“The advances in tools that we and others have made in precisely measuring and manipulating neurons and neural circuits now make it possible to read and write brain activity at the same time, and communicate with the brain in the fast timescale on which it operates,” says Stanley, professor in the Wallace H. Coulter Department of Biomedical Engineering. “We think this is a game-changer, experimentally and computationally.”
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