If the drug proves viable to treat epilepsy in humans, it would be the first to provide lasting relief from seizures even after patients stopped taking it. iStock/cagkansayin

A drug typically prescribed for arthritis halts brain-damaging seizures in mice that have a condition like epilepsy, according to researchers at the University of Wisconsin–Madison.

The drug, called tofacitinib, also restores short-term and working memory lost to epilepsy in the mice and reduces inflammation in the brain caused by the disease. If the drug proves viable for human patients, it would be the first to provide lasting relief from seizures even after they stopped taking it.

“It ticks all the boxes of everything we’ve been looking for,” says Avtar Roopra, a neuroscience professor in the UW–Madison School of Medicine and Public Health and senior author of the study, which was published last week in the journal Science Translational Medicine.

Epilepsy is one of the most common neurological diseases, afflicting more than 50 million people around the world. While there are many known causes, the disease often appears after an injury to the brain, like a physical impact or a stroke.

Some days, months or even years after the injury, the brain loses the ability to calm its own activity. Normally balanced electrical activity through the brain goes haywire.

“The system revs up until all the neurons are firing all the time, synchronously,” says Roopra. “That’s a seizure that can cause massive cell death.”

And the seizures repeat, often at random intervals, forever. Some drugs have been useful in addressing seizure symptoms, protecting patients from some of the rampant inflammation and memory loss, but one-third of epilepsy patients do not respond to any known drugs, according to Olivia Hoffman, lead author of the study and a postdoctoral researcher in Roopra’s lab. The only way to stop the most damaging seizures has been to remove a piece of the brain where disruptive activity starts.

On their way to identifying tofacitinib’s potential in epilepsy, Hoffman and co-authors used relatively new data science methods to sift through the way thousands of genes were expressed in millions of cells in the brains of mice with and without epilepsy. They found a protein called STAT3, key to a cell signaling pathway called JAK, at the center of activity in the seizure-affected mouse brains.

“When we did a similar analysis of data from brain tissue removed from humans with epilepsy, we found that was also driven by STAT3,” Hoffman says.

“[The discovery] ticks all the boxes of everything we’ve been looking for,” says Avtar Roopra (right), seated with research collaborator Olivia Hoffman.

Meanwhile, Hoffman had unearthed a study of tens of thousands of arthritis patients in Taiwan aimed at describing other diseases associated with arthritis. It turns out, epilepsy was much more common among those arthritis patients than people without arthritis — but surprisingly less common than normal for the arthritis patients who had been taking anti-inflammatory drugs for more than five-and-a-half years.

“If you’ve had rheumatoid arthritis for that long, your doctor has probably put you on what’s called a JAK-inhibitor, a drug that’s targeting this signaling pathway we’re thinking is really important in epilepsy,” Hoffman says.

The UW researchers ran a trial with their mice, dosing them with the JAK-inhibitor tofacitinib following the administration of a brain-damaging drug that puts them on the road to repeated seizures. Nothing happened. The mice still developed epilepsy like human patients.

Remember, though, that epilepsy doesn’t often present right after a brain-damaging event. It can take years. In the lab mice, there’s usually a lull of weeks of relatively normal time between the brain damage and what the researchers call “reignition” of seizures. If it’s not really epilepsy until reignition, what if they tried the drug then? They devised a 10-day course of tofacitinib to start when the mouse brains fell out of their lull and back into the chaos of seizures.

“Honestly, I didn’t think it was going to work,” Hoffman says. “But we believe that initial event sort of primes this pathway in the brain for trouble. And when we stepped in at that reignition point, the animals responded.”

The drug worked better than they could have imagined. After treatment, the mice stayed seizure-free for two months, according to the paper. Collaborators at Tufts University and Emory University tried the drug with their own mouse models of slightly different versions of epilepsy and got the same, seizure-free results.

Roopra’s lab has since followed mice that were seizure-free for four and five months. And their working memory returned.

“These animals are having many seizures a day. They cannot navigate mazes. Behaviorally, they are bereft. They can’t behave like normal mice, just like humans who have chronic epilepsy have deficits in learning and memory and problems with everyday tasks,” Roopra says. “We gave them that drug, and the seizures disappear. But their cognition also comes back online, which is astounding. The drug appears to be working on multiple brain systems simultaneously to bring everything under control, as compared to other drugs, which only try to force one component back into control.”

Because tofacitinib is already FDA-approved as safe for human use for arthritis, the path from animal studies to human trials may be shorter than it would be for a brand-new drug. Roopra’s epilepsy research has long been funded by the National Institutes of Health as well as key early support from Madison-based Lily’s Fund for Epilepsy Research and CURE Epilepsy.

The next steps toward human patients largely await NIH review of new studies, which have been paused indefinitely amid changes at the agency.

For now, the researchers are focused on trying to identify which types of brain cells are shifted back to healthy behavior by tofacitinib and on animal studies of even more of the many types of epilepsy. Hoffman and Roopra have also filed for a patent on the use of the drug in epilepsy.

This research was funded in part by grants from the National Institutes of Health (R01NS108756, R21NS093364, R01NS112308, NS112350, R01NS105628, R01NS102937 and R21NS120868).

Heart muscle cells grown from human induced pluripotent stem cells (in green) have successfully integrated into rhesus macaque heart muscle in this microscope image of heart tissue from a new study by UW–Madison and Mayo Clinic researchers. Photo courtesy Emborg Lab / UW–Madison

Heart muscle cells grown from stem cells show promise in monkeys with a heart problem that typically results from a heart defect sometimes present at birth in humans, according to new research from the University of Wisconsin–Madison and Mayo Clinic.

Heart disease, the No. 1 killer of Americans, can affect people at any time across their lifespans — even from birth, when heart conditions are known as congenital heart defects. Regenerating tissue to support healthy heart function could keep many of those hearts beating stronger and longer, and this is where stem cell research is stepping in.

A research team led by Marina Emborg, professor of medical physics in the UW–Madison School of Medicine and Public Health, and Timothy Nelson, physician scientist at the Mayo Clinic in Rochester, Minnesota, reported recently in the journal Cell Transplantation that heart muscle cells grown from induced pluripotent stem cells can integrate into the hearts of monkeys with a state of pressure overload.

Also referred to as right ventricular dysfunction, pressure overload often affects children with congenital heart defects. Patients experience chest discomfort, breathlessness, palpitations and body swelling, and can develop a weakened heart. The condition can be fatal if left untreated.

Nearly all single ventricle congenital heart defects, particularly those in the right ventricle, eventually lead to heart failure. Surgery to correct the defect is a temporary solution, according to the researchers. Eventually, patients may require a heart transplant. However, the availability of donor hearts — complicated by the young age at which most patients require a transplant — is extremely limited.

In their new study, the researchers focused on grafts of stem cell-derived cardiomyocytes as a possible complementary treatment to traditional surgical repair of cardiac defects. Their goal was to directly support ventricular function and overall healing.

“There is a great need for alternative treatments of this condition,” says Jodi Scholz, the study’s lead author and chair of Comparative Medicine at Mayo Clinic. “Stem cell treatments could someday delay or even prevent the need for heart transplants.”

The researchers transplanted clinical-grade human induced pluripotent stem cells — cells collected from human donors, coaxed back into a stem cell state and then developed into cell types compatible with heart muscle — into rhesus macaque monkeys with surgically induced right ventricular pressure overload. The cells successfully integrated into the organization of the surrounding host myocardium, the muscular layer of the heart. The animals’ hearts and overall health were closely monitored throughout the process. The authors noted that episodes of ventricular tachycardia (an increased heart rate) occurred in five out of 16 animals receiving transplanted cells, with two monkeys presenting incessant tachycardia. These episodes resolved within 19 days.

“We delivered the cells to support existing cardiac tissue,” Emborg says. “Our goal with this particular study, as a precursor to human studies, was to make sure that the transplanted cells were safe and would successfully integrate with the organization of the surrounding tissue. We leveraged my team’s experience with stem cells and cardiac evaluation in Parkinson’s disease to assess this innovative therapeutic approach.”

The research proved the feasibility and safety of using stem cells in the first nonhuman primate model of right ventricular pressure overload. Macaques, in particular, have been critical to advancing stem cell therapies for heart disease, kidney disease, Parkinson’s disease, eye diseases and more.

“The demonstration of successful integration and maturation of the cells into a compromised heart is a promising step towards the clinical application for congenital heart defects,” Emborg says.

The research was supported by the Todd and Karen Wanek Family Program for Hypoplastic Left Heart Syndrome and National Institutes of Health Grant P51OD011106 to the Wisconsin National Primate Research Center.

Parkinson’s disease damages neurons in the brain that produce dopamine, a brain chemical that transmits signals between nerve cells. The disrupted signals make it progressively harder to coordinate even simple movements and cause rigidity, slowness and tremors that are the disease’s hallmark symptoms. Patients are typically treated with drugs like L-DOPA to increase dopamine production. Although the drugs help many patients, they present complications and lose their effectiveness over time.

Researchers at the Wisconsin National Primate Research Center successfully grafted brain cells called dopaminergic neuronal progenitor cells into the brains of cynomolgus macaque monkeys. California-based Aspen Neuroscience provided the cells, grown from multiple lines of human induced pluripotent stem cells, along with key pieces of the equipment for delivering them to specific parts of the brain.

Ed Wirth

“By the time of diagnosis, it is common for people with Parkinson’s to have lost the majority of dopaminergic neurons, leading to progressive loss of motor and neurological function,” explains Edward Wirth III, an expert in cell therapies, study co-author and Aspen’s chief medical officer. “To replace these lost cells, we must target a very specific area of the brain with a high degree of surgical precision. Utilizing the latest advances in intraoperative MRI guided techniques, the patient’s new cells are transplanted, a few microliters at a time, to the exact area where they are most needed.”

Working with potential cell therapies in pursuing treatments for Parkinson’s disease is a particular specialty of the team at Marina Emborg’s lab and their primate center colleagues.

Marina Emborg

“Using autologous cells, a patient’s own cells, avoids the need to use immunosuppression to keep the patient’s body from rejecting or attacking the graft,” says Emborg, a UW–Madison professor of medical physics. “Aspen has developed the technological methods for manufacturing, for quality control, that makes it feasible at scale to make autologous cells and get them to the patients.”

The researchers’ results in non-human primates, which supported Aspen’s successful Investigational New Drug application to the Food and Drug Administration to begin human trials, were published today in the Journal of Neurosurgery.

Andrés Bratt-Leal

“This study was an important step in our work to bring the promise of a cell-replacement therapy to people with Parkinson’s disease,” says Andrés Bratt-Leal, study co-author, Aspen Neuroscience co-founder and senior vice-president of research and development. “The results were instrumental in opening our first-in-human trial and informing how we deliver patients’ own cells to them in the study.”

The UW–Madison scientists, led by Parkinson’s researcher Emborg, took up the Aspen-funded work fresh off their own success (published in 2021) reversing Parkinson’s symptoms in monkeys by grafting neurons grown from the monkeys’ own cells, called an autologous transplant.

The 2021 study, using cells grown by stem cell researcher Su-Chun Zhang of the UW–Madison School of Medicine and Public Health, added new dopamine-producing neurons to each animal’s brain through injections guided in real time by MRI to an area of the brain called the putamen. Dopamine production increased dramatically, as did the monkeys’ motor skills. At the same time, symptoms of depression and anxiety were reduced.

The new study was designed to test the delivery of Aspen’s human cells. Wirth and Aspen scientists worked with Emborg’s team to bridge the monkey-to-human application. While Emborg’s previous study administered cells to the putamen through the top of the skull, the Aspen study examined cell administration through the back of the skull — an angle that could allow surgeons to reach their target with fewer insertions of the apparatus that delivers the new cells into the brain.

“The core idea is to decrease the risk of infection, the trauma, the surgical time the patient spends under anesthesia,” Emborg says. “The fewer tracks you have to follow through the brain, the better for all of that.”

Six monkeys received grafts of the human neurons through two paths in each side, or hemisphere, of their brains, with more cells deposited on one side of the brain than the other. A control group of three animals underwent the procedure without the cell delivery.

“In tissue samples taken seven and 30 days after the procedures, we found the grafted cells persisted in five of the animals,” Emborg says.

The researchers confirmed the presence of Aspen’s human neurons in the monkeys’ brains, finding more cells in the hemispheres that were injected with a higher dose, more cells in the 30-day tissue samples compared to the seven-day samples and the presence of a protein produced by young neurons working to integrate with neighboring cells — all signs the cells grafts were successful.

It was a true collaboration, according to Emborg — between the Aspen scientists, her lab and the Wisconsin National Primate Research Center veterinarians and staff — to validate the company’s procedures and equipment before study co-author Paul Larson, a neurosurgeon at Banner – University Medical Center Tucson and professor of neurosurgery at the University of Arizona College of Medicine – Tucson, began Aspen’s first-in-human trial with people with Parkinson’s in April.

The work done to refine the logistics, surgical equipment and techniques in the animal procedures will inform the way patients in the human trial receive and recover from the new therapy, providing hope for those struggling with a debilitating disease.

“Our results were all so exciting,” Emborg says. “And then, when I saw they had been able to begin with a human patient this spring, I just had tears in my eyes.”

This research was supported by Aspen Neuroscience and the National Institutes of Health (grant No. P51OD011106).

​​​​ Illustration: Beth Atkinson

Size doesn’t matter when it comes to genome sequencing in the animal kingdom, as a team of researchers at the Morgridge Institute for Research recently illustrated when assembling the sequences for two new reference genomes — one from the world’s largest mammal and one from one of the smallest.

The blue whale genome was published in the journal Molecular Biology and Evolution, and the Etruscan shrew genome was published in the journal Scientific Data.

Research models using animal cell cultures can help navigate big biological questions, but these tools are only useful when following the right map.

“The genome is a blueprint of an organism,” says Yury Bukhman, first author of the published research and a computational biologist in the Ron Stewart Computational Group at the Morgridge Institute, an independent research organization that works in affiliation with the University of Wisconsin–Madison in emerging fields such as regenerative biology, metabolism, virology and biomedical imaging. “In order to manipulate cell cultures or measure things like gene expression, you need to know the genome of the species — it makes more research possible.”

The Morgridge team’s interest in the blue whale and the Etruscan shrew began with research on the biological mechanisms behind the “developmental clock” from James Thomson, emeritus director of regenerative biology at Morgridge and longtime professor of cell and regenerative biology in the UW School of Medicine and Public Health.  It’s generally understood that larger organisms take longer to develop from a fertilized egg to a full-grown adult than smaller creatures, but the reason why remains unknown.

“It’s important just for fundamental biological knowledge from that perspective. How do you build such a large animal? How can it function?” says Bukhman.

Bukhman suggests that a practical application of this knowledge is in the emerging area of stem cell-based therapies. To heal an injury, stem cells must differentiate into specialized cell types of the relevant organ or tissue. The speed of this process is controlled by some of the same molecular mechanisms that underlie the developmental clock.

What genomes from animals of different sizes can tell us about our own health

Understanding the genomes of the largest and smallest of mammals may also help unravel the biomedical mystery known as Peto’s paradox. This is a curious phenomenon in which large mammals such as whales and elephants live longer and are less likely to develop cancer — often caused by DNA replication errors that occasionally happen during cell division — despite having a greater number of cells (and therefore more cell divisions) than smaller mammals like humans or mice.

Meanwhile, knowledge of the Etruscan shrew genome will enable new insights in the field of metabolism. The shrew has an extremely high surface to volume ratio and fast metabolic rate. These high energy demands are a product of its tiny size — no bigger than a human thumb and weighing less than a penny — making it an interesting model to better understand regulation of metabolism.

The blue whale and Etruscan shrew genome projects are part of a large collaborative effort involving dozens of contributors from institutions across North America and several European countries, in conjunction with the Vertebrate Genomes Project.

The mission of the VGP is to assemble high-quality reference genomes for all living vertebrate species on Earth. This international consortium of researchers includes top experts in genome assembly and curation.

“The VGP has established a set of methods and criteria for producing a reference genome,” Bukhman says. “Accuracy, contiguity, and completeness are three measures of quality.”

Previous methods to sequence genomes used short read technologies, which produce short lengths of the DNA sequence 150 to 300 base pairs long, called reads. Overlapping reads are then assembled into longer contiguous sequences, called contigs.

Contigs assembled from short reads tend to be relatively small compared to mammalian chromosomes. As a result, draft genomes reconstructed from such contigs tend to be very fragmented and have a lot of gaps.

Instead, the team used long read sequencing, with reads around 10,000 base pairs in length, with the principal advantage being longer contigs and fewer gaps.

“Then you can use other methods such as optical mapping and Hi-C to assemble contigs into bigger structures called scaffolds, and those can be as big as an entire chromosome,” Bukhman explains.

The researchers also analyzed segmental duplications, large regions of duplicated sequence that often contain genes and can provide insight into evolutionary processes when compared to other species, either closely or distantly related.

They found that the blue whale had a large burst of segmental duplications in the recent past, with larger numbers of copies than the bottlenose dolphin and the vaquita (the world’s smallest cetacean, the order of mammals including whales, dolphins and porpoises). While most of the copies of genes created this way are likely non-functional, or their function is still unknown, the team did identify several known genes.

One encodes the protein metallothionein, which is known to bind heavy metals and sequester their toxicity — a useful mechanism for large animals that accumulate heavy metals while living in the ocean.

How reference genomes can help with wildlife conservation

A reference genome is also useful for species conservation. The blue whale was hunted almost to extinction in the first half of the 20th century. It is now protected by an international treaty and the populations are recovering.

“In the world’s oceans, the blue whale is basically everywhere except for the high Arctic. So, if you have a reference genome, then you can make comparisons and can better understand the population structure of the different blue whale groups in different parts of the globe,” Bukhman says. “The blue whale genome is highly heterozygous, there’s still a lot of genetic diversity, which has important implications for conservation.”

Which begs the question: how do you go about acquiring samples from a large, endangered creature that exists in the vastness of the oceans?

“The logistics posed several challenges, including the fact that blue whale sightings in our area are very rare and almost unpredictable,” says Susanne Meyer, a research specialist at the University of California Santa Barbara, who spent over a year to coordinate the permits, personnel and resources needed to procure the samples.

Once their local whale-watching team determined the timing and coordinates of the whale sightings, they brought in licensed whale researcher Jeff K. Jacobsen to perform the whale biopsies using an approved standard cetacean skin biopsy technique, which involves a custom stainless steel biopsy tube fitted to a crossbow arrow.

The team acquired samples from four blue whales, which Meyer used to develop and expand fibroblasts in cell culture for the genome sequencing and further research use.

Size doesn’t matter when it comes to an animal’s genome

While the Etruscan shrew genome wasn’t studied as extensively as the blue whale genome, the team reported an interesting finding.

“We found that there are relatively few segmental duplications in the shrew genome,” Bukhman says, while emphasizing that this result does not necessarily correlate to the diminutive size of the shrew itself. “While shrews belong to a different mammalian order, some similarly small rodents have lots of segmental duplications, and the house mouse is kind of a champion in that sense that it has the most. So, it’s not a matter of size.”

As the Vertebrate Genomes Project makes strides in producing more high-quality reference genomes for all vertebrates, Bukhman is hopeful that contributions to those efforts will continue to advance biological research in the future.

These studies were supported by grants from the National Science Foundation (2046753, DBI2003635, DBI2146026, IIS2211598, DMS2151678, CMMI1825941 and MCB1925643) and National Institutes of Health (R01GM133840).

Learn more here.

Ting Fu

Inflammation in the gut can trigger a doom loop of sorts. The condition messes with the sensitive relationships between food, digestive acids, microbes and the immune system in ways that can promote further inflammation and, sometimes, the eventual growth of tumors.

Scientists at the University of Wisconsin–Madison have identified a promising new target for treatments that could help the millions of people worldwide who suffer from inflammatory bowel disease and related colorectal cancers.

An essential regulator of gut health

Under the guidance of Ting Fu, an assistant professor in the UW–Madison School of Pharmacy, researchers uncovered a previously unknown function of a protein that is central to gut health and implicated in the development of colitis, a severe and chronic form of IBD. A debilitating condition in and of itself, colitis is also linked to an increased risk for colorectal cancer. The team’s findings suggest that the protein is a promising target for future colitis treatments.

The protein is called the farnesoid X receptor, or FXR. It helps control the production of bile acids that digest fats. Working in tandem, FXR and bile acids play several critical roles in maintaining a healthy gut. Together, they help balance gut bacteria, promote a healthy intestinal lining, and influence immune cells called macrophages that patrol the digestive system and ward off pathogens that sneak in with the food we eat.

“This balance can be thrown off when FXR isn’t functioning properly,” says Xingchen Dong, a postdoctoral researcher in Fu’s lab and the study’s lead author.

Dong, Fu and their colleagues studied mice with chronic gastrointestinal inflammation that led to the growth of tumors in their colons. This mimicked the effects of colitis-associated colon cancer in humans. They found that FXR was not functioning properly in these mice, messing with the signals the protein sends to manage bile acids.

At the same time, they noted shifts in the chemistry of bile acids within the animals’ gastrointestinal tracts. These changes affected both “host” bile acids, produced by the mice themselves, and microbial bile acids, which are the product of gut microbes metabolizing host bile acids.

In a cascade of negative effects, the altered bile acids prompted changes in the behavior of gut macrophages, leading to a large increase in certain proteins called cytokines that promote inflammation. This observation provided compelling new evidence for how FXR dysfunction changes the behavior of gut macrophages, initiating the inflammation doom loop that can cause colitis and eventually lead to aggressive cancers.

From a scientific perspective, “it is exciting to see that gut macrophages have the capability to sense both host and microbial bile acids and exhibit diverse responses to various bile acids, which leads to changes in their state or activity,” says Fu.

This image shows the effect of pro-inflammatory cytokines on the growth of a mouse intestinal organoid. Intestinal stem cells (red) are indicative of inflammation and Ki67 proteins (green) are associated with tumors. Photo courtesy of Ting Fu

A promising treatment for colitis and associated cancers

FXR dysfunction is implicated in a number of gastrointestinal diseases, and Fu’s team investigated whether existing drugs aimed at activating FXR — called FXR agonists — might prove effective treatments for colitis and associated colon cancers.

Mice treated with either of two FXR agonists — fexaramine D or FDA-approved obeticholic acid — saw marked improvement in FXR functioning, with a stream of other positive effects, including rebalanced bile acids, improved macrophage function and reduced intestinal inflammation.

Colorectal tumors in the treated mice were also “profoundly reduced” in both number and size, according to Fu. The median survival time of mice with colitis-associated cancer was twice as long in animals treated with the compounds compared to those that received no treatment. The findings were published Jan. 23 in the journal JCI Insight.

“This study shows that FXR plays a crucial role in regulating how macrophages behave in the gut,” says Fu. “This could be really important for developing new treatments for IBD and colitis-associated cancers.”

Fu intends to continue exploring compounds that promote FXR function as potential treatments for colitis and related cancers, though any treatment strategies for human patients based on this research will require further exploration and confirmation.

This work was funded by UW–Madison startup grants (AAI3795, AAI3894), UW Carbone Cancer Center startup support (AAI5122), UW Center for Human Genomics & Precision Medicine startup support (AAI5319), fall competition support from Wisconsin Alumni Research Foundation (AAL8735), as well as American Cancer Society institutional grant (AAH4826), and American Cancer Society – Coaches vs. Cancer – Bo Ryan-Jay Holliday Families FundResearch Scholar Grants (RSG-23-1150338-01) and the Margaret Q. Landenberger Research Foundation.

This image of a section through the midface of a mouse embryo illustrates fusion of the tissues that form the secondary palate above the tongue. Green staining illustrates cells expressing a key enzyme that mediates DNA methylation, blue indicates nuclei of all cells, red indicates epithelial cells.

Cleft lip and palate are the most common craniofacial birth defects in humans, affecting more than 175,000 newborns around the world each year. Yet despite decades of research, it’s still not known what causes most cases or what can be done to prevent them. But a recent study from the University of Wisconsin School of Veterinary Medicine (SVM) has uncovered new information about orofacial development in mice that researchers believe could one day help reduce the risk of these birth defects in humans.

Published this week in the Proceedings of the National Academy of Sciences (PNAS) the study provides the first direct evidence of a mechanism called DNA methylation being required for craniofacial development. DNA methylation is a process where a group of molecules are added to DNA that change the expression of genes without actually altering the DNA sequence. It’s also affected by various environmental factors. The researchers discovered that disruption to DNA methylation interferes with development of the lip and palate and causes these birth defects in mice.

Rob Lipinski

Led by Robert Lipinski, associate professor of comparative biosciences at the UW School of Veterinary Medicine, the research is an important step toward developing preventive strategies that could one day lessen the risk of cleft lip and palate, known collectively as orofacial clefts (OFCs), in both animals and humans.

“We knew from past research that genetics and the environment interact to cause these types of birth defects, but our understanding of the environmental component lagged far behind that of genetics.” says Lipinski. “Unlike genetics, we don’t have a permanent record of the prenatal environment that can be examined retrospectively, but connecting OFCs to DNA methylation helps narrow our focus on the particular environmental influences that modify the risk for these types of birth defects.”

His team’s work confirmed the essential role of DNA methylation in regulating orofacial development during embryonic development and demonstrates how disruptions to that process alter the ability of stem cells to form the connective tissue of craniofacial bone and cartilage, resulting in OFCs.

Lipinski and his team arrived at these results by first genetically manipulating DNA methylation in two separate groups of mouse embryos. The experiments resulted in seemingly contradictory results, with OFCs developing in one group of mice, but not the other. To understand why there was a difference between the groups, the team conducted another round of experiments in which they inhibited DNA methylation in mouse embryos at different stages of development. The timing of when DNA methylation occurs was critical to the development of orofacial clefts.

They found that exposure on the 10th gestational day resulted in OFCs but administering the same inhibitor just 48 hours later resulted in normal orofacial development.

Identifying this narrow window of gestational sensitivity is important, Lipinski says, because it not only helps narrow the focus of the next stage of his team’s research but it will also help design future public education initiatives once more is known about the modifiable environmental and behavioral risk factors that impact OFC risk in humans.

The 10th gestational day in mouse embryos corresponds with the beginning of the 5th week of embryonic development in humans–a stage at which many pregnancies may not yet be recognized.

“We know DNA methylation can be influenced by a variety of environmental factors, including maternal stress, diet, and exposure to drugs, toxins and environmental pollutants, and having a better understanding how orofacial development is regulated by environmentally sensitive mechanisms could directly inform birth defect prevention strategies,” he says. “This next phase of our team’s research is focused on identifying specific factors that influence DNA methylation during orofacial development and which could therefore alter OFC risk.”

Lipinski and his team are uniquely positioned to pursue this next stage of research because of another important outcome of the study: a new in vitro model the team developed. The model will allow them to rapidly screen thousands of dietary and environmental factors in a laboratory dish before testing the impact of specific factors on cleft susceptibility in mouse models.

The results in cell and animal models will help the researchers more quickly and accurately identify factors likely to be of consequence to human development.

Orofacial clefts of the upper lip and palate affect approximately 1 in 700 newborns, and individuals with OFCs navigate feeding difficulties as infants that require multiple surgeries, dental procedures, and speech therapy during childhood and adolescence. Studies have shown higher mortality rates at all stages of life for individuals with OFCs.

This study was supported by funding from the National Institutes of Health under award numbers R03DE027162, R56DE030917, RO1DE032710, U01 DK11807, and R01DK099328, and T32ES007015. Additional support was also provided by the University of Wisconsin Hilldale Undergraduate Research Award.

Researchers at the University of Wisconsin–Madison have taken a novel approach to assessing canine vision. Their recent study uses a dog’s interest in a variety of video content to better measure the quality of its vision. iStock / damedeeso

Ever wonder what kind of TV shows your dog might choose if they could work the remote control? New research from the University of Wisconsin–Madison’s School of Veterinary Medicine provides some answers, but the study was more interested in solving a longstanding problem in veterinary medicine than turning canine companions into couch potatoes.

According to Freya Mowat, veterinary ophthalmologist and professor in the School of Veterinary Medicine’s department of surgical sciences, researchers wanted to determine factors, including age and vision, that influence a dog’s interest in interacting with video content. Ultimately, the goal of the study, which launched two years ago, was to support development of more sensitive ways to assess canine vision — something that has been sorely lacking in veterinary medicine.

Freya Mowat

“The method we currently use to assess vision in dogs is a very low bar. In humans, it would be equivalent to saying yes or no if a person was blind,” says Mowat. “We need more sensitive ways to assess vision in dogs, using a dog eye chart equivalent. We speculate that videos have the potential for sustaining a dog’s attention long enough to assess visual function, but we didn’t know what type of content is most engaging and appealing to dogs.”

Published recently in the journal Applied Animal Behaviour Science, the study found that dogs are most engaged when watching videos that feature other animals. Content featuring other dogs was the most popular. But if a National Geographic documentary about canine evolution seems too highbrow for your four-legged friend, Scooby Doo might be a perfectly acceptable option as well.

To better understand the type of content dogs might be most attracted to on screen, Mowat created a web-based questionnaire for dog owners around the globe to report the TV-watching habits of their canine companions.

Participants responded to questions about the types of screens in their homes, how their dogs interacted with screens, the kinds of content their dogs interacted with the most, as well as information about their dog’s age, sex, breed and where they live. They also provided descriptions of their dogs’ behavior when watching videos. Most commonly, dog owners described their pets’ behavior as active — including running, jumping, tracking action on screen and vocalizing — compared with passive behaviors like lying down or sitting. Dog owners also had the option to show their dog(s) four short videos featuring subjects of possible interest, including a panther, a dog, a bird and traffic moving along a road. They were then asked to rate their dog’s interest in each video and how closely the dog tracked the moving objects on the screen.

In this video, a dog watches scenes of another dog going for a walk.

Mowat received 1,600 responses from dog owners across the world, including from the United States, Canada, the United Kingdom, the European Union and Australasia. Of those respondents, 1,246 ultimately completed the study. The following are some of the most interesting highlights:

• Age and vision were related to how much a dog interacted with a screen.
• Sporting and herding dog breeds appear to watch all content more than other breeds.
• Video content featuring animals was the most popular, with other dogs being by far the most engaging subjects to watch.
• Humans do not appear to be very appealing for dogs to watch, ranking ninth out of 17 predetermined categories.
• Cartoons were engaging for more than 10% of dogs.
• Movement on screens was a strong motivator for screen attention.

Mowat says she plans to build on the results of this study. Future research will focus on the development and optimization of video-based methods that can assess changes in visual attention as dogs age as well as answer questions that could help our four-legged friends age as gracefully as possible.

“We know that poor vision negatively impacts quality of life in older people, but the effect of aging and vision changes in dogs is largely unknown because we can’t accurately assess it,” she says. “Like people, dogs are living longer, and we want to make sure we support a healthier life for them as well.”

Another goal for Mowat is to compare how a dogs’ vision ages compared with the human or humans they share a home with.

“Dogs have a much shorter lifespan than their owner, of course, and if there are emerging environmental or lifestyle factors that influence visual aging, it might well show up in our dogs decades before it shows up in us,” she explains. “Our dogs could be our sentinels — the canine in the proverbial coal mine.”

This study was supported in part by an NIH career development grant to Mowat (K08EY028628), a Companion Animal Fund Grant from the UW–Madison School of Veterinary Medicine, a grant from Research to Prevent Blindness, Inc. to the UW–Madison Department of Ophthalmology and Visual Sciences and a core grant for Vision Research from the NIH to UW–Madison (P30 EY016665).