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At the University of South Florida’s Hyperbaric Biomedical Research Laboratory, ongoing work to combat oxygen toxicity seizures in Navy divers has expanded to include research that may lead to non-toxic cancer therapies combining dietary supplements and hyperbaric oxygen.

Jay Dean, PhD, professor in the Department of Molecular Pharmacology and Physiology, USF Health Morsani College of Medicine, created and has directed the collaborative research facility since it opened in 2006.  The laboratory houses chambers that can mimic the adverse environments of high atmospheric pressure (hyperbaric) experienced by deep-sea divers. With instrumentation specially designed to operate under extreme pressures, Dr. Dean and his colleagues can analyze the molecular responses of cells as well as the physiological changes in animal models exposed to changing concentrations of oxygen, nitrogen and other gases.

Jay Dean, PhD, professor of molecular pharmacology and physiology, established and directs the USF Hyperbaric Biomedical Research Laboratory.

To date, Dr. Dean and his USF colleague, Dominic D’Agostino, PhD, have adapted electrophysiology, radio-telemetry and various types of microscopy techniques for use under hyperbaric pressures, including fluorescence, confocal and atomic force microscopy.

“Atomic force microscopes are common, but not atomic force microscopes placed under hyperbaric pressure,” said Dr. Dean, one of the world’s leading experts in hyperbaric neurophysiology. “We’ve been able to successfully apply very powerful research tools to these unique conditions.” 

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Probing oxygen toxicity’s role in seizures

In the last decade Dr. Dean’s laboratory, sponsored by the Office of Naval Research Undersea Medicine Program, has helped shed light on the role of hyperbaric oxygen toxicity in triggering seizures. The condition can be a life-threatening by-product of breathing too much oxygen at high ambient pressures that impacts deep-sea divers as they swim deeper and longer.

Navy SEALs are especially at risk because they wear a closed circuit rebreather, to mitigate the narcotic and other debilitating effects of nitrogen and carbon dioxide breathed under increasing ocean pressure. The special device filters out these gases in such a way that bubbles do not appear on the water’s surface – useful in helping avoid enemy detection. However, the additional stealth comes at a cost. The ratio of oxygen the divers breathe greatly increases the deeper they plunge (essentially becoming pure oxygen) and, when combined with physical exertion and mission stress, can lead to nausea, dizziness, seizures, and even coma or death – all symptoms of oxygen toxicity.

Deep-sea divers can be at risk for oxygen toxicity seizures, a life-threatening condition caused by breathing too much oxygen at high ambient pressures. – U.S. Department of Defense photo

A possible countermeasure, anti-seizure sedatives, requires high doses that could impair warfighters’ mental and physical performance.

Without a reliable way to treat oxygen toxicity or predict which divers are more prone to seizures than others, the Navy takes rigorous precautions to restrict all divers to no more than 10 minutes in 50 feet of seawater.

“This risk of central nervous system oxygen toxicity limits oxygen’s use — not only in diving operations, but also its clinical applications in hyperbaric oxygen therapy,” Dr. Dean said.

Hyperbaric oxygen therapy, which increases blood oxygen to temporarily restore blood gases and tissue function, can help treat unhealed wounds, burns, crushing injuries, decompression sickness, carbon monoxide poisoning, and other medical conditions. The therapeutic benefit might be maximized if the doses of hyperbaric oxygen administered could be boosted without the risk of central nervous system oxygen toxicity.

In their search to find solutions, Dr. Dean and colleagues analyze the response of individual brain cells to the powerful effects of oxygen and other gases under altered pressure. In the laboratory’s hyperbaric chambers, they measure changes in brain cell membranes and electrical activity, and the damage of oxygen-induced free radicals.

An intracellular recording of the electrical signaling by a brain cell (middle trace) in a rodent brain slice that is stimulated by hyperbaric oxygen (top trace).

The researchers also monitor physiological changes in the breathing and heart rate of normal rats moving about in a chamber mimicking the environment of an increasingly deep dive. An electroencephalogram (EEG) shows electrical signals in the brain in real time, indicating the hyperexcitability that precedes and peaks with oxygen toxicity seizures.

Promising discoveries to predict, delay seizures

The USF group has made what could be a key discovery – the breathing rate of the rats exposed to pure oxygen increases several minutes before a seizure starts. “This may be a biomarker – a physiological signal that predicts the impending seizure,” Dr. Dean said.

If this early-predictor hypothesis bears out in larger animal models, Dr. Dean said, the next step would be to work with the Navy to devise and test a mask-fitted with a device designed to monitor divers’ breathing underwater. The ultimate aim: preventing oxygen-induced seizures to safely allow Navy SEALs to dive deeper and longer.

Another of the laboratory’s major findings evolved from an idea by Dr. Dean’s former postdoctoral fellow, Dominic D’Agostino, PhD, to harness the power of ketones, natural compounds produced by the body when it burns fat during periods of fasting or calorie restriction.

Dominic D’Agostino, PhD, associate professor of molecular pharmacology and physiology who collaborates with Dr. Dean, has expanded the laboratory’s research to include metabolic therapeutics. His group is investigating the combination of the ketogenic diet and/or ketone supplements with hyperbaric oxygen as a potential non-toxic cancer therapy.

Now an associate professor of molecular pharmacology and physiology, Dr. D’Agostino has focused on better understanding how the ketogenic diet — a special low-carbohydrate, high-fat diet that elevates blood ketones — produces anticonvulsive and neuroprotective effects. And, more recently he has worked with collaborators in academia and industry to develop and test naturally derived and synthetic supplements to boost blood ketones to mimic the ketogenic diet’s therapeutic effects.

Successfully used by physicians to treat drug-resistant epilepsy or other seizure disorders, the ketogenic diet shifts the brain’s energy source from glucose toward using ketones as a super fuel. However, it takes several days, or event weeks, for the body to adapt to this change in brain energy metabolism. That limitation and other problems associated with adhering to such a strict low-carbohydrate diet make nutritional ketosis less than ideal for Navy SEALs on a mission.

“The big advantage of putting the diet in a pill or liquid form is that you can achieve therapeutic ketosis in 30 minutes, instead of a week,” Dr. D’Agostino said.

A microscopic image of neurons hyper-excited by exposure to pure oxygen under high pressure in the hyperbaric chamber.

In a first of its kind study, Dr. D’Agostino tested whether feeding laboratory rats ketone esters and placing them in the hyperbaric chamber simulating underwater conditions could delay oxygen toxicity seizures. It worked.

More research is needed, but the experiments pave the way for a ketone supplement that would allow Navy SEALS to dive longer while protecting them against seizures, Dr. Dean said. “If what we’ve observed in rat model experiments holds true in humans, the Navy diver should be able to increase the amount of time spent at a depth of 50 feet of seawater (10 minutes) by 600 percent… which means that the divers could get more work done with fewer dives.”

“When the brain is running off ketones, it becomes much more resilient in terms of preserving brain energy and preventing a seizure,” Dr. D’Agostino said.

Based on research led by Dr. D’Agostino, USF has several patents pending for producing brain metabolism-enhancing ketone supplements, which may have a broad range of applications for neurodegenerative diseases like Alzheimer’s and ALS, diabetes and certain cancers as well as seizure disorders – all associated with impairments in metabolic regulation.

Earlier this year USF hosted the first international conference drawing doctors and researchers to discuss the effects of nutritional ketosis and metabolic therapeutics on the treatment of various diseases.

Stephanie Ciarlone, MS, a doctoral student in the USF Health Byrd Alzheimer’s Institute Laboratory of Edwin Weeber, PhD, (seated) professor of molecular pharmacology and physiology. Ciarlone’s preclinical studies of ketone esters in an Angelman syndrome mouse model are helping lay the foundation for what may be the first clinical trial of a USF-developed ketone ester in children with the rare neurogenetic disorder. — Photo by Sandra C. Roa, USF Health Communications

Among the presenters was Stephanie Ciarlone, MS, a doctoral student in the USF Health Byrd Alzheimer’s Institute laboratory of Edwin Weeber, PhD, where her research focuses on treatment options for Angelman syndrome, including ketone esters. This rare neuro-genetic disorder affects young children who commonly suffer debilitating drug-resistant seizures as the condition worsens.

With Dr. D’Agostino as a collaborator, a recent study by Ciarlone found that ketone supplements, without dietary restriction, delayed the onset of seizures and reduced the their number by 50 percent in a mouse model of Angelman syndrome. The ketone esters also improved learning and memory and motor coordination in the mice.

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Dr. Weeber, professor of molecular pharmacology and physiology, is working with Dr. D’Agostino to move their preclinical studies to the first clinical trial of a USF-developed ketone ester in children with Angelman syndrome. The study is expected to begin within a year.

From neuroprotection to exploring non-toxic cancer therapy

A serendipitous thing happened while Dr. D’Agostino and Angela Poff, PhD, research associate, were studying the neuroprotective effects of ketone supplements in different cell models. While examining cancer cells under a microsope specially designed to withstand the barometric pressure in the hyperbaric chamber, they observed that these cells were selectively vulnerable to high pressure oxygen at levels not harmful to healthy cells. They also noticed that the cancer cells did not proliferate when put in a petri dish with ketone supplements as a fuel source.

Cancer cells exhibit altered metabolic processes that could potentially be exploited to shut down their proliferation and survival. Solid tumors have areas of low oxygen, or hypoxia, that actually help promote a cancer’s aggressive growth. “So, the idea was that if we put more oxygen into the blood, which is what the hyperbaric oxygen chamber does, it will diffuse further into the tissue and help shut down areas promoting the tumor growth,” Dr. Poff said.

Angela Poff, PhD, research associate, led a study targeting cancer metabolism with hyperbaric oxygen and ketosis.

In addition, cancer cells use carbohydrate-derived glucose to generate most of their energy, so some research suggests a ketogenic diet that rigorously limits carbohydrates may help slow cancer’s growth, Dr. Poff said.

Armed with this two-pronged approach, the researchers embarked on their first cancer experiments in the Hyperbaric Lab. They discovered that combining hyperbaric oxygen and ketosis reduced the proliferation of metastatic cancer cells. Then, moving their research to a mouse model for aggressive metastatic cancer, they showed that combining a ketogenic diet and ketone supplements with hyperbaric oxygen therapy slowed tumor growth and doubled the survival time of the rodents. Their study was published online last year in PLOS ONE and the theory behind this approach was highlighted in an article in Carcinogenesis.

Hyperbaric oxygen by itself only slightly inhibited the spread of cancer in the mice. “But when we combined hyperbaric oxygen with ketosis induced by the ketogenic diet and our ketone ester, the potent synergistic effect was greater than the individual therapies alone,” Dr. Poff said. In particular, adding the ketone ester to the mix of the ketogenic diet and hyperbaric oxygen boosted the anti-cancer effects.

Images of rat brain slices used to study how hyperbaric oxygen disrupts brain cell function to cause seizures.

Next, the USF researchers say they will work on fine-tuning the combination therapy – finding what doses of ketone supplementation and levels of oxygen work to optimize the anti-cancer effects.

While more research is needed, Dr. D’Agostino said, “this combination therapy could represent a non-toxic strategy to help metabolically manage cancer and enhance the effectiveness of standard cancer treatment with chemotherapy and radiation.”

Dr. D’Agostino and Dr. Poff

The USF Hyperbaric Biomedical Research Laboratory houses various pressure chambers, including a 3.2-ton one specially designed for use with an atomic force microscope, which mimic the extreme environmental conditions challenging deep-sea divers.

The USF Hyperbaric Laboratory, including an interview with Dr. Dean, will be included in an upcoming independent documentary on nitrogen narcosis, a major limiting factor in the performance of deep-sea divers. The video will feature Sherri Ferguson of Simon Fraser University in British Columbia, who studies the health effects of narcosis in divers.

Video and photos by Katy Hennig, USF Health Office of Communications 

USF Health PhD graduate Justin Trotter leaves this weekend for Stanford University School of Medicine in Palo Alto, CA, where he will work for a recent Nobel Prize winner in medicine.  Trotter’s postdoctoral fellowship will play out in the large laboratory of Stanford neuroscience researcher Thomas Sudhof, MD, a Howard Hughes Medical Institute investigator who just last week jointly won the 2013 Nobel Prize in Physiology or Medicine for insights into the traffic control system for living cells.

Justin Trotter, PhD, (left) USF Health neuroscience graduate, with major professor Edwin Weeber, PhD, chief scientific officer of the USF Health Byrd Alzheimer’s Institute.

“That just doesn’t happen very often,” said Edwin Weeber, PhD, professor of molecular pharmacology and physiology and chief scientific officer of the USF Health Byrd Alzheimer’s Institute.   Dr. Weeber was Trotter’s major professor while the young neuroscientist completed his doctoral studies in neuroscience at the Byrd Institute.

“Justin is the most driven graduate student I’ve had the pleasure to mentor,” Dr. Weeber said. “His ability to gain a fellowship with a Nobel Prize winner in one of the country’s top laboratories shows that USF and the Byrd Institute are training the next generation of scientists whose research will make a real difference.”

Trotter successfully defended his doctoral dissertation “Cellular and Molecular Mechanisms of Reelin Signaling in the Adult Hippocampus” on Sept. 27.  His doctoral research focused on signaling pathways important in brain development and their role in molecular mechanisms that give rise to learning and memory and that may be disrupted by Alzheimer’s disease.

“When I spoke to Tom (Sudhof) to endorse Justin’s application to his laboratory, I told him that Justin was the most brilliant young scientist I had ever met,” said Joachim Herz, MD, of the Department of Molecular Genetics, University of Texas Southwestern Medical Center, who served as the external chair for Trotter’s dissertation committee.

“My only concern and advice to Tom was that he should cut his travels short in the future, otherwise he might find Justin running the laboratory upon his return.”

2013 Nobel Prize-winning neuroscience researcher Thomas Sudof, MD

Probing the neurobiology of learning and memory

For a person to think, move, feel or remember, the neurons in that person’s brain must communicate across junctions known as synapses. Increasing evidence has linked impairments in synaptic transmission to diseases such as Alzheimer’s and autism.

Working with Dr. Sudhof’s team, Trotter will still study how nerve cells communicate with one another to precisely exchange information across synapses within millisecond timescales. But at Stanford he will focus on the role of signaling pathways in autism instead of Alzheimer’s.

Trotter was offered postdoctoral fellowships at three of the country’s leading research institutions — the National Institutes of Health Eunice Kennedy Schriver National Institute of Child Health and Human Development, the Gladstone Institute affiliated with the University of California San Francisco, and, his first choice, Stanford University

He interviewed with Dr. Sudhof and 15 of his postdoctoral fellows July 19 and was offered the position by Dr. Sudhof  himself a week later, well before the Nobel Prize announcement.

“When I saw that he was on a team that made the finalists, I thought wouldn’t it be fun if he actually won,” Trotter said. “Then, the awards committee made the announcement the next day, and people were congratulating me – but I didn’t really do anything.”

Early fascination with science cultivated on a fish farm

Trotter grew up in Palm Bay, FL, but spent most weekends working on a tropical fish farm in nearby Fellsmere, where his father and grandfather operated the acquaculture business. He attributes his early fascination with science to what he learned on the fish farm, including how to breed and care for African cichlids.

In elementary school, while many classmates relied on their parents for help with science fair projects, Trotter looked forward to the challenge of creating and carrying out his own experiments.

“Science projects became my means of self-expression,” said Trotter, who won many regional and state science fair awards throughout middle and high school.  “I enjoyed designing experiments to test assumptions and garner facts about the natural world.”

By 10^th grade he worked his way into an independent research project at Florida Institute of Technology, where he studied the molecular biology of starfish fertilization.  During one late-night experiment, Trotter said, he accidently hit his hand on a glass pipette filled with mercury. A hospital X-ray showed the shattered glass (from the broken pipette) scattered around a joint where the mercury injected.

“After about a week my hand was swollen and I had to get it operated on,” he said. “To this day I still have a small black, metallic circle near the injection site… Fortunately the type and quantity of mercury I was exposed to poses no danger.”

By 11^th grade Trotter was testing algae extracts from the Indian River Lagoon and the Antarctica for anti-cancer properties.  He even set up a temporary lab culture room at his house when the laboratory space where he worked was taken over by scientists preparing experiments to accompany a Columbia space shuttle flight.

“I needed to determine whether the extracts that I had prepared possessed the ability to slow down the division rate of leukemia cells,” he said. “Needless to say, my mom learned to avoid wondering what I was doing in the den.”

Passion for neuroscience nurtured at USF

Trotter has spent the last five years at USF, where he says his passion for neuroscience was nurtured. He earned a bachelor of science degree in biomedical sciences here, followed by a master’s of science and PhD degrees in medical sciences, both with a concentration in neuroscience.

Along the way he has co-authored 15 journal articles with Dr. Weeber and/or other USF faculty members, including Lynn Martin, PhD, of the Department of Integrative Biology.  He was a member of an interdisciplinary team awarded a highly competitive 2012-13 USF Graduate Student Research Challenge Grant.  Dr. Weeber and Trotter also have a patent pending for a new therapeutic approach for treating brain injuries.

At the USF Health Byrd Alzheimer’s Institute, Trotter found in Dr. Weeber a mentor who shared his passion for delving into how synapses work with the hope that the research will lead to future treatments for Alzheimer’s disease or other cognitive disorders.

“I’ve really enjoyed the collaborative spirit at the Byrd Institute,” Trotter said.  “They’ve provided me with the resources and support needed to move forward in the development of my scientific career.”

And this USF graduate continues to move ahead — taking his place next month in the laboratory of a new Nobel Prize winner.  “It’s an amazing opportunity,” Trotter said.

– Photos by Eric Younghans, USF Health Communications

For families battling Angelman Syndrome, the small clinical trial at USF Health is a first step toward finding a treatment for the severe neurological disorder

For Peyton Elkins, 6, who has a rare neuro-genetic disorder called Angelman Syndrome (AS), every day is a challenge.  She cannot walk without help. She can’t dress or feed herself. She can’t talk, so her parents cannot always tell what’s upsetting her when she cries.
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“I have hope, because we can’t live any other way,” said Peyton’s mother Melissa Elkins, of Knoxville, TN.  “Science is making big strides.  I know there will be a cure one day, and maybe before that there will be some treatments to help her with her challenges.”

Clockwise from left, Jim and Melissa Elkins with daughter Peyton, who has the rare neurogenetic disorder Angelman Syndrome, and Peyton’s younger siblings Ella and Owen.

Peyton is one of 24 children nationwide selected to participate in a clinical trial at USF Health investigating whether off-label use of the FDA-approved antibiotic minocycline will help improve the symptoms of AS. Currently, there is no cure or treatment for the disorder, which affects about one in every 15,000 children.

Over the course of their enrollment in the 16-week single-arm, open label trial, each child undergoes a series of tests, blood work and behavioral evaluations.  Families travel to Tampa, FL, three separate times as part of their commitment to the study, which is funded through a grant by the Foundation for Angelman Syndrome Therapeutics (FAST).  

The drug being studied was tested first in mice genetically altered to have AS. Although the condition is caused by a single or inactive gene, it results in relatively widespread and severe developmental delays affecting motor function, thinking and behavior. Seizures are one of the most common and debilitating conditions affecting children with the syndrome.

“If minocycline does have an effect on sleep, on seizures, on cognitive ability, it will potentially give us more insight into what we need to be looking at in other therapeutics, so it could open the door to a lot of different treatments for these kids,” said trial principal investigator Edwin Weeber, PhD, chief scientific officer and a neuroscientist at the USF Health Byrd Alzheimer’s Institute.

Supported in part by FAST, Weeber heads one of a handful of laboratories nationwide probing the molecular mechanisms underlying AS in a mouse model for the disorder and exploring strategies that may rapidly yield effective treatments. 

Studies using an Angelman Syndrome mouse model, conducted in Dr. Edwin Weeber’s laboratory at the USF Health Byrd Alzheimer’s Institute, helped fast track one of the first clinical trials for AS.

One promising approach has been to test the effectiveness of existing FDA-approved drugs in treating the cognitive, motor coordination and physiological impairment established in the AS mouse model. Minocycline, a drug traditionally used to treat bacterial infections, has minimal side effects, penetrates the brain and appears to work in a way that may counter the molecular defects observed in AS, Weeber said.

When Weeber’s team administered minocycline to AS mice, the treated mice performed nearly the same as typical mice on tests for motor coordination and their ability to learn and remember increased as well. The USF preclinical research helped fast track minocycline as a candidate for one of the few clinical trials for AS.

Peyton was diagnosed at age 9 months, when her seizures began. Her parents suspected something was not right a couple of months earlier when their baby had trouble holding up her head and sitting. But, the symptoms of AS can mimic other neuro-developmental disorders like cerebral palsy and autism, so a quick blood test is required for genetic confirmation of the syndrome.

 Over the last few years, Peyton’s parents have done all they can to provide supportive care to improve their daughter’s quality of life – from physical, occupational and speech therapy to medications to ease seizures and sleep difficulties.

 And, Melissa Elkins says, the family, which includes Peyton’s siblings Owen, 4, and Ella, 1, draws emotional support from the tight-knit community of families touched by AS.  Many parents connect through online social networking sites like the one maintained by FAST.

Peyton, 6, is enrolled in a national clinical trial at USF Health testing whether the FDA-approved antibiotic minocycline can alleviate Angelman Syndrome’s debilitating symptoms.

While she realizes the small minocycline clinical trial is just a first step, Elkins is excited to be part the journey toward a treatment.

 “When you have a baby, you have huge dreams for them.  Then you get a diagnosis and those dreams are shattered.  I’m hoping we can have those back,” Elkins said. “I continue to hope that my child Peyton can one day be as typical as my other two.” 

 Results from the minocycline trial, which ends in February, are expected in early 2013, Weeber said. “If we see positive effects in these children, it would lead to a larger clinical trial.

 More information on Angelman Syndrome research, please visit http://www.weeberlab.com/clinical_trials.html or www.CureAngelman.org

– Photos courtesy of Melissa Elkins. Photo of Dr. Weeber by Eric Younghans/USF Health Communications

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