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STAT

An MUSC blog
Keyword: neuroscience

A pericytePictured Above: A fluorescent pericyte cell body (red) with processes
extending along adjacent capillaries (green). Courtesy of Dr. Andy Shih and Robert Underly of the Medical University of South Carolina.

 

Pericytes are the primary locus of matrix-mellaproteinase-9-dependent (MMP-9) capillary damage and blood leakage during ischemia, according to preclinical findings reported by Medical University of South Carolina (MUSC) investigators in an article published online on November 14, 2016 by The Journal of Neuroscience. In vivo two-photon microscopy revealed MMP-9 activity and plasma leakage disproportionately occurred at locations where pericyte somata were attached to the endothelium. These results suggest that pericytes, normally essential for blood-brain barrier (BBB) function, contribute to capillary damage during stroke.

The BBB—a highly specialized vascular structure—prevents the entry of blood-borne substances that can harm the brain (e.g., neurotransmitters such as glutamate, clotting factors such as fibrin, and free radical–generating substances such as iron). During ischemic stroke and related cerebrovascular diseases, the BBB is damaged, allowing incursion of blood plasma that injures neurons and other structures essential for normal cerebral function. 

The role of pericytes as builders and custodians of the BBB is well recognized, but how pericytes respond to blood flow loss in the adult brain has largely been a mystery, until now.

MUSC researchers recently harnessed cutting-edge technology to image pericytes in the intact brains of live mice and to spatially and temporally track the proteolytic enzyme, MMP-9, as the BBB degraded and blood began leaking into the brain.

Findings from this novel study not only provide critical new information about pericytes as a potent source of MMP-9 during BBB leakage but also open new discovery pathways for future therapies in neurological conditions involving ischemia.

It all began when Robert Underly, a PhD candidate in the MUSC College of Graduate Studies Neuroscience Program and first author on the article, noticed that, when a laser was used to induce ischemic strokes in the laboratory, BBB leakage occurred at very specific sites along the capillaries. "I'd assumed that blood leakage occurred along the entire capillary length,” said Underly. “But it wasn't like that. There were hotspots that leaked first and more than the rest of the capillary bed. That was really unexpected."

Andy Y. Shih, Ph.D., Assistant Professor of Neurosciences and senior author on the article, and his team followed up on Underly's observation.  "We found a very close association between where the round cell bodies of pericytes were located and where the leaks occurred,” said Shih. “So that was our first clue that the pericytes were possibly doing something harmful in the early stages of an ischemic stroke."

It is well known that the BBB becomes dysfunctional when genetic defects disrupt pericyte-endothelial signaling from birth. However, very little is known about how normally developed pericytes in the adult brain respond during acute ischemia, and only one or two studies have investigated this in vivo.

"Pericytes have a lot of potential functions—they seem to be a sort of a jack-of-all-trades,” said Shih. “We've had an idea of what these cells do, but we haven't been able to visualize and track them in vivo until recently."

 

The team was also intrigued by a handful of published studies showing that various inflammatory signaling cascades can induce pericyte MMP-9 expression.

"The problem is that, like pericytes, MMPs are hard to study in vivo—most of what we know about them is from studies on cultured cells or extracted brains,” said Underly. “We wanted to probe this process in live animals so we could see the spatiotemporal relationship between pericytes and MMP activation in vivo—in the acute stroke time frame."

To do this, the team combined several novel tools to create a unique study protocol using transgenic mice, two-photon fluorescent microscopy, and a fluorescent gelatin-based reporter of MMPs that only one other research group had ever used to study the intact brain.

The researchers also used photothrombosis to block blood flow in a small area of the capillary bed and imaged transgenic mouse lines expressing bright fluorescent reporters specifically in the pericytes to clearly identify them.

"The successful combination of technologies is certainly one of the innovations of this project,” said Shih. “It's the first study to combine these tools to look at the relationship among pericytes, MMP activity, and BBB leakage in ischemia."

Their findings revealed that ischemia resulted in extremely rapid (within tens of minutes), localized activation of MMP-9 and plasma leakage. Furthermore, plasma leakage occurred preferentially where the pericyte somata adjoined the capillary wall—not homogenously along the length of the capillaries as previously imagined by the group. These results provide strong evidence that pericytes—normally protectors of the BBB—contribute to early BBB degradation during ischemic stroke.

Using the new technology, the team did not have to extract and cut the brain and so did not lose the structure of the vasculature and blood flow.

“We had an intact system and could see where things were coming from and we were very surprised,” said Shih. “I thought, 'I've been looking at this for years and I never knew that there was this beautiful co-localization.' It told us we were looking at something really interesting. The pericytes seem to nurture or damage the BBB depending on the conditions they're put in."

This discovery opens many directions for further study and could eventually lead to new therapeutic options for patients experiencing an acute stroke.

"The very rapid reaction we saw to ischemia is really important and provides clues to potential mechanisms by which MMPs may be up-regulated,” explained Underly. “This is a future direction for our research—to define upstream regulators of this process that can be therapeutically targeted."

"The findings raise so many new research questions,” said Shih. “For example, why do pericytes have so much MMP? What are they doing with it? What happens to pericytes days to weeks after an ischemic event? There's so much still to be understood about the acute phenomena—we're focused on that for now. In the future, we could look at later, post-injury, events to see what happens next in the life of the pericyte."

Indeed, in vivo cellular-level imaging research has a bright future.

"There's a renaissance happening with the development of new molecular tools to image and modify cell function in vivo," said Shih. "We're going to see a lot more integration between tool-makers and in vivo imaging groups in the next decade or two. There are going to be many more studies looking at the intact brain."

"It's important to fund projects like this because with in vivo imaging we're able to track exactly what happens when brain function breaks down,” said Underly. “The disease state occurs in front of our eyes."

Stock-Image-of-Brain

Researchers at the Medical University of South Carolina (MUSC) have used viruses to infect neurons with genes that allow them to switch on brain receptors involved in suppressing addiction relapse. Results of these preclinical studies were published in the September 28th, 2016 issue of the Journal of Neuroscience. The technology, called designer receptors exclusively activated by designer drugs, or DREADDs, is one of the most promising gene therapies for the future treatment of addiction in humans.

 The brains of people who use cocaine become hijacked by drug cues. Powerful memories are formed between these cues–such as the using environment and drug paraphernalia–and the dopamine flood that occurs from using the drug itself. In users trying to quit, these drug cues activate an intense desire to seek cocaine again.

 Resistance to relapse is partly mediated in the ventromedial prefrontal cortex–the brain region slightly above and behind our eyes, where previously learned associations are broken. This region of the brain stores extinction memory, which works to suppress the emotional response to drug cues, according to Jamie Peters, Ph.D., Research Assistant Professor in the MUSC Department of Neuroscience.

 “Extinction doesn’t overwrite the original memory,” explained Peters. “It just helps suppress the pathological component of the response.” 

 Peters and her colleague Peter W. Kalivas, Ph.D., Chair of the MUSC Department of Neuroscience, wanted to know if the response to drug cues associated with the dopamine rush of cocaine could be suppressed when the extinction memory region was activated. To test their hypothesis, they obtained viruses carrying the DREADD gene from Bryan L. Roth, M.D., Ph.D., in the Department of Pharmacology at the University of North Carolina Chapel Hill. The DREADD technology is openly accessible to researchers around the world through the National Institutes of Mental Health Psychoactive Drug Screening Program, where Roth serves as director.

 The viruses work by inserting the DREADD gene directly into the genome of cells, causing them to grow receptors on their surface that are normal except for a slight alteration. These receptors express a protein encoded by the DREADD gene that allows them to be activated by a single drug designed to bind that protein. In this case, the Peters lab infused a virus carrying a DREADD gene designed to change surface receptors on neurons. After the neurons were infected, they would fire in response to administration of the designer drug. Because the body’s other cells had not been infected with the DREADD gene, they would remain unaffected.

 “This new approach for treating drug addiction is exactly what is needed because it is targeted to a specific circuit in the brain regulating addiction,” said Kalivas. “This may allow the circuit to be selectively regulated with minimum side effects on other circuits and brain functions.”

 The researchers allowed rats to self-administer cocaine by pressing one of two levers, one active and one inactive. Once a rat pressed the active lever, cocaine was delivered along with a brief audio tone and a pulse of light that would serve as the drug cues. After a series of daily cocaine exposure sessions, the rats had learned to associate the simple drug cues with cocaine availability. Then they were removed from the drug. Next a surgical technician infused virus carrying the DREADD gene directly into the rats’ ventromedial prefrontal cortices. After two weeks of cocaine abstinence, the rats were placed back in front of the two levers in ten daily sessions, but this time the levers produced neither cues nor cocaine. The next day, rats were subjected to a relapse test where the cues were returned. Before testing, half of the rats were given designer drug and half were not. Next, rats underwent an additional relapse test where they were given a low dose of cocaine to trigger relapse.

 The experiments worked. Rats that were given the designer drug relapsed less in the presence of drug reminder cues. However, when exposed to cocaine again, rats relapsed regardless of whether they were given the designer drug. In other words, Peters’ hypothesis was correct: rats with activated extinction memories weren’t as susceptible to relapse triggered by cocaine-associated cues but were still vulnerable when exposed to cocaine again. This meant that extinction memory retrieval reduced relapse triggered by reminder cues.

 This study shows that it is possible to use this technology to target a small population of cells in the brain that is important for regulating addiction, thereby inhibiting the drive to relapse to addictive drug use. In the future, Peters hopes that safe and effective viruses of this kind can be infused into the brains of human addicts during neurosurgery. A person would simply take a pill to activate the extinction memory region of their brain, helping them to suppress the urge to seek out drug in the face of those reminder cues. Since extinction memory isn’t as powerful as the emotional response to a drug, this strategy could work when paired with effective psychological counseling approaches such as cognitive behavioral therapy.

Clinicians interested in using DREADDs in humans will have to remain patient, however. DREADDs have to be designed to match drugs that suppress only memories of drug cues while leaving other memories unaffected. And the crystal structure of newer human-appropriate designer drugs bound with the special receptors is being actively investigated in order to visualize exactly how they might work some day in patients with cocaine addiction.

 “Certainly within my lifetime I would expect to see these virus-mediated gene therapies start to be used in the brain, in a neurosurgical setting,” said Peters. “You can envision a person ultimately taking a pill to activate this very specific part of his or her brain.”

Image licensed from iStock.com.

Predicting speech fluency after stroke. Brain images showign features of damage to grey-matter and white-matter regions of brain, reflecting their importance in predicting speech fluency.

Image Caption: Predicting speech fluency after stroke. These are features of gray-matter cortical regions (left) and white-matter tracts (right), reflecting their importance in predicting speech fluency scores. Regions/connections are marked in red when they strongly influence speech fluency, in blue when their influence is moderate, and are left uncolored when the influence is weak or non-existent. Image used courtesy of Dr. Leonardo Bonilha and Dr. Grigori Yourganov of the Medical University of South Carolina, who own the copyright for the image. Published in the June 22 issue of the Journal of Neuroscience (doi:10.1523/JNEUROSCI.4396-15.2016).

Loss or impairment of the ability to speak is one of the most feared complications of stroke—one faced by about 20% of stroke patients. Language, as one of the most complex functions of the brain, is not seated in a single brain region but involves connections between many regions.

In an article published in the June 22, 2016 issue of the Journal of Neuroscience (doi:10.1523/JNEUROSCI.4396-15.2016), investigators at the Medical University of South Carolina (MUSC) and the University of South Carolina (USC) report that mapping all of the brain’s white matter connections after stroke, in addition to imaging the areas of cortical tissue damage, could better predict which patients will have language deficits and how severe those deficits will be. The totality of the brain’s connections is referred to as the connectome.

“Imaging the connectome of patients after stroke enables the identification of individual signatures of brain organization that can be used to predict the nature and severity of language deficits and one day could be used to guide therapy,” said MUSC Health neurologist Leonardo Bonilha M.D., Ph.D., senior author on the Journal of Neuroscience article, whose laboratory focuses on connectome imaging, particularly as it relates to language loss after stroke. Grigori Yourganov, Ph.D., is the first author on the article. Julius Fridriksson, Ph.D., Chris Rorden, Ph.D., and  Ezequiel Gleichgerrcht, Ph.D, aphasia researchers at USC who recently received NIH funding to establish a Center for the Study of Aphasia Recovery and who are long-time collaborators of the Bonilha laboratory, are also authors on the article.

This study is the one of the first to use whole-brain connectome imaging to examine how disruptions to white matter connectivity after stroke affect language abilities. White matter fiber tracts are the insulated wires that connect one area of the brain to others. White matter is named for the myelin sheaths (insulation) that cover the many axons (wires) that make up the fiber tracts.

“If you have two brain areas and both of them have to work together in order to carry out a function and the stroke lesion takes out axons that connect those brain areas—the two areas are intact but the communication between them is disrupted and so there is dysfunction,” said Yourganov.

Currently, structural magnetic resonance imaging (MRI) is used after stroke to assess lesions in the cortical tissue—the brain’s grey matter. However, the extent of cortical damage often does not correlate with the severity of language deficits.

“Stroke patients sometimes have significant impairments beyond the amount of cortical damage,” said Bonilha. “It is also hard to predict how well a patient will recover based on the cortical lesion alone.”

Could connectome-based imaging be a useful complement for assessing damage to the brain’s connections after stroke and for guiding rehabilitative therapy?

The study led by Bonilha took an important first step toward answering these questions. The study, which enrolled 90 patients at MUSC and USC with aphasia due to a single stroke occurring no less than six months prior, assessed four areas related to speech/language using the Western Aphasia Battery—speech fluency, auditory comprehension, speech repetition, and oral naming—as well as a summary score of overall aphasia. Within two days of behavior assessment, each of the patients underwent imaging studies—both T1- and T2- weighted MRI, typically used after stroke to map cortical damage, and diffusion imaging, used for connectome mapping. The team then used a type of machine learning algorithm—support vector regression (SVR)—to analyze the imaging results and make predictions about each patient’s language deficits.  In essence, an algorithm was created that could derive the WAB score from either a feature relevant to imaging of the grey matter damage by structural MRI or a feature relevant to connectome imaging of the brain’s white matter fiber tracts. The team used 89 of the 90 patients as training sets for SVR and then used the algorithm to predict language defect/preservation in the 90th patient. This was done for each of the 90 patients and, in each patient, for both features identified via structural MRI and connectome imaging.

The accuracy of the algorithm’s prediction of WAB score for each patient was then assessed by comparing it to the WAB score determined via behavioral testing. Connectome-based analysis was as accurate as cortical lesion mapping for predicting WAB scores. In fact, it was better at predicting auditory comprehension scores than was lesion-based imaging using structural MRI and only slightly less accurate at predicting speech fluency, speech repetition, and naming scores.

The study demonstrates that damage to the white matter fiber tracts that connect the brain’s regions plays a role beyond cortical damage in language impairment after stroke. Furthermore, this study also discloses that connections in the brain’s parietal region are particularly important for language function, especially fluency. This region is less likely to sustain damage after stroke, even in patients who experience aphasia, suggesting that damage or preservation of the brain’s connections in this region could play a key role in determining who will experience aphasia and who will have the best chances for recovery. The integrity of these connections could not be mapped with conventional structural MRI but can now be assessed through connectome-based analysis.

The study findings also suggest that connectome-based analysis could be used to inform a more individualized approach to stroke care. Because the algorithms developed using these study patients as the training set are generalizable to a broader stroke population, connectome-based analysis could one day be used to identify the distinctive features of each patient’s stroke—which connections have been lost and which preserved—and then the algorithm could be used to predict the type and severity of language impairment and the potential for recovery. This information could then be used to direct rehabilitative therapy to improve outcomes. 

“By mapping much more accurately the individual pattern of brain structural connectivity in a stroke survivor, we can determine the integrity of neuronal networks and better understand what was lesioned and how that relates to language abilities that are lost,” said Bonilha. “This is, broadly stated, a measure of post-stroke brain health. It is the individual signature pattern that could also be used to inform about the personalized potential for recovery with therapy and guide treatments to focus on the deficient components of the network.”

the Sinu-lok device

The Medical University of South Carolina’s Institute for Applied Neurosciences (IAN), a technology accelerator that was created in 2013 to develop neuroscience technologies, has licensed its first medical device.  Amendia, based in Marietta, Georgia, has acquired the exclusive worldwide rights to manufacture and sell Sinu-Lok™, a rod implant used in minimally invasive lumbar spinal fusion surgery. In this procedure, the screws that will connect the rod implant are extra tall to allow for smaller incisions when putting them in. Today’s standard rod implant is slightly bowed. When the surgeon tightens the construct down, the rod’s curvature forces the top part of the tall screws to bump together or even overlap. This puts stress on the construct components, which can lead to a loosening of the construct after the surgery and other complications.

Alternatively, the Sinu-Lok rod has a smooth oscillating shape that provides several concave locations in which the screws can seat when tightened. This patented shape also provides an extended range of axial connections between the screw-rod interface when the construct is tightened, creating a divergence of the screw towers instead of the convergence caused by the standard rod.

The licensing of Sinu-Lok is a key milestone for IAN, says Ted Bird, Chief Development Officer for IAN. “This license validates our unique technology acceleration model and demonstrates our ability to develop, patent, and commercialize valuable health care ideas from MUSC. Sinu-Lok was the first product developed and patented by IAN and we are very pleased to have a commercial partner like Amendia that is committed to manufacturing and commercializing this product as soon as possible to benefit surgeons and ultimately patients.”

IAN has seven additional active projects in the areas of concussion detection, neurovascular cranial access systems, brain tumors, spine surgery, intra-operative neuro-monitoring, and general surgical devices, as well as a current pipeline of 10 potential projects being reviewed.

Illustration by Emma Vought

brain optics graphicIn 2015, the National Science Foundation's (NSF) Experimental Program to Stimulate Competitive Research (EPSCoR) focused on three priority areas: the complexity of the brain, clean energy, and food security. After reviewing nearly sixty applications, the NSF awarded eight awards to the tune of $42 million in early August. Only three of these awards were for brain research, and MUSC was a recipient. A $4 million grant to MUSC will be used to develop and implement new optical technologies to image brain function at a very high resolution. The Principal Investigators of the grant (Peter W. Kalivas, Ph.D., and Prakash Kara, Ph.D., both from the Department of Neurosciences at MUSC) felt that partnering with the University of Alabama at Birmingham (UAB) played an important role in securing this grant. UAB and MUSC will bring complementary technologies to develop a parallel pipeline of state-of-the-art brain scanners in both states. Optical technologies previously used to look at the stars in the sky will now be miniaturized to look inside the brain. Additional partners in this grant include Furman University, the University of South Carolina Beaufort, and Clemson University.

The NSF recognizes the importance of funding new research projects that merge science, technology, engineering, and mathematics (STEM). Grant recipients must also develop a STEM workforce in order to grow their research programs. While the NSF funds basic science research, this infrastructure often leads to new discoveries that improve health care. For example, this grant thematically focuses on developing tools to determine the precise mechanisms (on the microcircuit scale) by which neurons normally communicate with blood vessels in the brain. However, this “neurovascular” communication breaks down in many neurological diseases. Thus, this grant will have far-reaching impacts in the fields of neuroimaging, neurology, and neuroscience education.

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