Hydralazine Hydrochloride

Damage associated with Alzheimer's disease begins within hours after a concussion according to results from research that uses a 'mini-brain' lab on a chip device. How much time elapses between a blow to the head and the start of damage associated with Alzheimer's disease? A device that makes it possible to track the effects of concussive force on a functioning cluster of brain cells suggests the answer is in hours. The "traumatic brain injury (TBI) on a chip" being developed at Purdue University opens a window into a cause and effect that announces itself with the passage of decades but is exceedingly difficult to trace back to its origins. "We're basically creating a miniature brain that we can hit and then study," said Riyi Shi, lead researcher and the Mari Hulman George Endowed Professor of Applied Neuroscience in Purdue University's College of Veterinary Medicine. "We know there's a link between TBI and Alzheimer's; that's well established in clinical observation. But teasing out the basic essential pathway is not easy. With the TBI on a chip, we're able to test a lot of hypotheses that would be very difficult to do in living animals." In a study recently published in Lab on a Chip, a research team led by Shi subjected functioning clusters of cultured neurons from embryonic mice to three blows of 200 g-force, each approximating the higher end of what a football player receives in a single hit. The trauma leads to an immediate surge in production of acrolein -- a molecule associated with oxidative stress and neurodegenerative disease -- and a rise in misfolded clumps of the protein amyloid beta 42 (AB42), which is found in masses called plaques in the brains of people with Alzheimer's disease. Additional experiments traced the links between impact, acrolein and AB42. The device can also be used to test possible therapeutics, including drugs known to reduce acrolein levels. In the current study, Shi's team used the device to show that the drug hydralazine, a known acrolein scavenger that is approved by the U.S. Food and Drug Administration for lowering blood pressure, reduces the amount of acrolein and levels of misfolded AB42 produced in the cluster of neurons after a hit. Shi, who has a long history of studying neurodegenerative disease, acrolein and hydralazine, said the TBI on a chip enabled a finding he's sought over two decades of study. "Now that we know what's happening, is there something we can do about it? And the answer is yes," said Shi, who is also a member of the Purdue Institute for Integrative Neuroscience. "Acrolein is time-dependent; the longer it's there, the more AB42 aggregation it will cause. Here we show that if we lower acrolein with this drug, we can lower inflammation and AB42 aggregation." The device, custom-fabricated at the Purdue Center for Paralysis Research, uses a pendulum to deliver a specific g-force to a small chamber housing a cluster of a quarter million neurons supported by a bed of nutrients. A microelectronic array embedded in the chamber measures the electrical activity of the neurons, which will sustain functional firing patterns for several weeks, while a clear viewing port allows microscopic observation of the neurons. Researchers remove the cluster of neurons from the chamber at intervals to take specific biochemical measurements. "There's several unique things that we do here, but one of the biggest is that you can hit this chip without damaging it, so you can give an impact to a live model and continue to study it," Shi said. Shi began working on the device in graduate school, incorporating over the course of several decades features that make it possible to study the aftereffects of an initial blow. A 2022 paper in Nature Scientific Reports used the device to show the surge in acrolein that occurs after a hit, and Shi said the most recent findings hint at the power of the model. "Thanks to this device, people should know that when you get a concussion, you don't have 10 years before you will see damage," Shi said. "The clock starts ticking immediately, and if we want to do something about it, we need to act quickly." Within the first 24 hours after a hit, results show elevated levels of acrolein in the neuron clusters and a 350% increase in production of misfolded AB42. Shi said acrolein deforms normal AB42 by binding to sections of the protein that contribute to structural stability. Indeed, when the team conducted a simple experiment by combining large amounts of acrolein with normal purified AB42 suspended in fluid, they found elevated levels of misfolded AB42. The properly folded protein is sufficiently fragile that even subjecting normal purified AB42 in fluid (without acrolein) to an impact was enough to provoke misfolding. "This amyloid beta pathology started within hours, maybe immediately. That's never been heard of," Shi said. "It's like attacking the weight-bearing stud in a house wall. If you break that stud, of course the house is going to fall down." Shi was joined in the research by Purdue colleagues Edmond A. Rogers, first author, and co-authors Timothy Beauclair, Jhon Martinez, Shatha J. Mufti, David Kim, Siyuan Sun, Rachel L. Stingel, Nikita Krishnan and Jennifer Crodian, senior research associate at Purdue's Center for Paralysis Research, as well as Alexandra M. Dieterly of Charles River Laboratory. The study was supported by the state of Indiana, the National Institutes of Health and Plexon Inc. Moving forward, Shi said, he may be able to incorporate multiple additional features, which would allow the measurements of minute forces that cells experience during the blow, and biochemical testing -- like checking levels of acrolein -- without removing cells from the chamber.

Scientists have used Xenopus laevis tadpoles to tease out the genetic and biological mechanisms that enhance disease tolerance, and have identified drugs that can keep the tadpoles alive even in the presence of lethal bacteria. Many of the same mechanisms are also found in mammals, suggesting that humans and other animals could one day be treated for disease by increasing their tolerance to pathogens rather than killing the pathogens themselves. Why do some people seem to never get sick while others consistently fall prey to viruses and bacteria? How can the spouse of a sick person avoid catching their partner's bug despite sleeping next to them every night? Questions like these have become top-of-mind for many people during the COVID-19 pandemic, and scientists are now a big step closer to answering them thanks to some aquatic helpers: tadpoles. Researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University have discovered genetic and biological mechanisms that enhance disease tolerance -- the ability of cells and tissues to resist damage in the presence of invading pathogens -- in developing tadpoles of Xenopus laevis frogs, and identified drugs that can keep the tadpoles alive even in the presence of lethal bacteria. Many of the same mechanisms are also found in mammals, suggesting that infections in humans and other animals could one day be treated by increasing their tolerance to pathogens. "The standard approach to treating infections for the last 75 years has been to focus on killing the pathogen, but the overuse of antibiotics in livestock and in humans has led to the emergence of antibiotic-resistant bacteria that we are having a harder and harder time killing. Our research has shown that focusing on modifying a host's response to a pathogen rather than killing the pathogen itself could be an effective way to prevent death and disease without exacerbating the problem of antibiotic resistance," said first author Megan Sperry, Ph.D., a Postdoctoral Fellow at the Wyss Institute who is co-mentored by Wyss faculty members Michael Levin, Ph.D. and Donald Ingber, M.D., Ph.D. The research is published today in Advanced Science. Mapping the tadpole tolerance network The phenomenon of some hosts being tolerant of infectious pathogens that should sicken them has been well-documented in science over the last few decades. Mice, for example, can harbor pneumonia-causing Pneumococcus bacteria in their nasal passageswithout displaying signs of illness, and African and Asian monkeys are known to be less susceptible to certain pathogens than humans and our close ape relatives. Research into the biology of disease tolerance has found that it is associated with the activation of stress responses that are normally induced by a low level of oxygen (hypoxia). These cellular responses reprogram T cells, which reduces the amount of inflammation they cause, and also impact the movement of metal ions, which are crucial for bacterial survival. As part of the Wyss Institute's ongoing quest to identify drugs that could replicate these biological processes and induce tolerance in humans, Sperry and her team wanted to see if they could use a combination of computational techniques and hands-on experiments to tease out the genes and molecular pathways that control tolerance in Xenopus frogs, and then find existing drugs that could activate those pathways and induce a state of tolerance against pathogens. They chose to use Xenopus frog embryos for their studies because these embryos are easy to grow and analyze in large numbers and are known to display natural tolerance to high loads of certain types of bacteria. They exposed the embryos to six different species of pathogenic bacteria, and then analyzed the animals' gene expression patterns following infection. Embryos that encountered the more aggressive species Aeromonas hydrophila and Pseudomonas aeruginosa displayed visible changes in their physical development at 52 hours post-infection and widespread modifications to their gene expression patterns one day after infection, reflecting the animals' physiological responses to the pathogens. The other four species caused no visible change in the embryos, which at first suggested that the animals weren't reacting to the pathogens. But the genetic analysis told a different story. While two of the species, S. aureus and S. pneumoniae, caused very few genetic changes in the embryos' gene expression pro the species Acinetobacter baumanii and Klebsiella pneumoniae caused significant changes in a set of 20 genes that had been unchanged during infection with the more aggressive bacteria. These genetic changes seemed to correlate with a positive impact on the health of the developing frogs, implying that they could be involved in the animals' tolerance response. The researchers used a computational approach to map Xenopus genes that underwent significant change to their corresponding genes in humans, and analyzed how those genes interact with each other by organizing them into "gene networks." They found that the embryos that tolerated A. baumanii and K. pneumoniae had significant shifts in their gene networks that were distinct from the shifts seen in embryos that succumbed to infection with A. hydrophila and P. aeruginosa. One particular gene, HNF4A, was highly upregulated in the tolerant embryos, and was connected to several genes that are involved in transporting metal ions and increasing oxygen availability -- both processes that have been previously linked to disease tolerance. HNF4A also helps maintain the circadian rhythm, and the scientists found that flipping the embryos' light cycle increased tolerance against A. hydrophila infection, raising the intriguing possibility that modulating circadian rhythms could impact an organism's response to infection. "It was really exciting to see that pathogen tolerance seems to be modulated by multiple coordinated biological processes -- hypoxia, metal ion transport, and circadian rhythm -- because it might be possible to develop a whole class of drugs that simultaneously target multiple pathways to help make organisms more resistant to damage by infection while avoiding undesirable side effects," said co-author Richard Novak, Ph.D., a former Lead Staff Engineer at the Wyss Institute who is now co-founder and CEO of Unravel Biosciences. Treat the body, not the bug Armed with these promising results, Sperry, Novak, and their team set out to see if they could find any such drugs. First, they compared the gene expression signature they'd identified in the tolerant Xenopus embryos to existing data from mice and primates that had been infected with bacteria against which they were tolerant. They found that the gene networks in tolerant Xenopus embryos shared key overlaps with those found in tolerant mice and primates, and that twelve genes were common across all the species. Among those genes were several that are involved in a process called nuclear factor kappa B (NF-κB) signaling, which regulates inflammation responses to infection, as well as metal ion transport and cellular hypoxia responses. Confident that the Xenopus tolerance genes were a good proxy for aspects of tolerance in mammals, they then screened more than 30 pharmaceutical drugs that are known to impact metal ion transport or hypoxia by administering them to Xenopus embryos that were infected with A. hydrophila. Three drugs substantially increased embryo survival despite the presence of a pathogen that should have killed them: deferoxamine, an FDA-approved drug that binds to iron and aluminum ions; L-mimosine, which binds to iron and zinc; and hydralazine, which binds to metal ions and also dilates blood vessels. Because metal ion transport and hypoxia pathways are known to be interconnected, the researchers had a hunch that these metal scavenging drugs were stabilizing a biological protein called HIF-1α. HIF-1α regulates cells' responses to hypoxia and may be involved in reducing tissue damage and increasing disease tolerance. So, the scientists administered a drug called 1,4-DPCA, which is known to enhance the activity of HIF-1α via a separate but related mechanism. This drug increased Xenopus embryo survival to more than 80% in the presence of deadly bacteria. When the researchers added an inhibitor of HIF-1α along with 1,4-DPCA, the embryos succumbed to infection, confirming that HIF-1α is indeed a key player in infection tolerance. Crucially, the Xenopus genes that underwent the greatest changes in their expression levels due to treatment with 1,4-DPCA were also present in the 20-gene signature of pathogen tolerance that the researchers had identified previously, suggesting that the drug mimics aspects of natural tolerance including modulating genes involved in metal ion binding. "Ever since the germ theory of disease began to be accepted by science in the 19th century, treatment has focused on the pathogens themselves. But these experiments show that modulating a host's physiological responses to a pathogen deserves an equal amount of attention, and could offer a sorely needed alternative approach to treating disease," said co-author and Wyss Associate Faculty member Michael Levin, Ph.D., who is also the Vannevar Bush chair and Director of the Allen Discovery Center at Tufts University. However, the researchers caution that tolerance-inducing drugs are not a silver bullet against infections. Increasing humans' tolerance to infection could mean that they never fully clear a harmful pathogen from their bodies, which could have long-term health effects. In addition, these people with persistent, low-grade infections could spread the pathogen to others who are more susceptible. Therefore, drugs that increase tolerance are likely best used in combination with other measures like vaccines, or in isolated emergency situations such as protecting doctors and nurses who are responding to a deadly pathogen outbreak. "This is wonderful example of turning scientific or medical paradigms on their heads: rather than searching for yet another highly targeted antibiotic that pathogens will develop resistance to in the future, we chose to discover ways to stimulate the host to be tolerant to a broad range of infections. While this work is far from the clinic, it demonstrates the value of thinking outside the box, and opens up new approaches to therapeutics development," said senior author and Wyss Founding Director Donald Ingber, who also is the Judah Folkman Professor of Vascular Biology at Harvard Medical School (HMS) and Boston Children's Hospital, and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences. The team is continuing to investigate stress responses similar to those found in pathogen tolerance in Xenopus, now largely in the context of the Wyss Institute's Biostasis project. Additional authors of the paper include former Wyss Institute members Vishal Keshari, Alexandre Dinis, and Diogo Camacho, Ph.D.; Mark Cartwright, Ph.D., a Senior Scientist at the Wyss Institute; Jean-François Paré Ph.D., from Queen's University, Ontario, Canada; and Michael Super, Ph.D., a Lead Staff Scientist at the Wyss Institute. This research was supported by the US Defense Advanced Research Projects Agency (DARPA) under contract W911NF-16-C-0050.