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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 

Science is full of precision and vigilance. But sometimes, there are subtleties that present themselves that get ignored, pushed aside for the drive to stay on task or to stick with the parameters of a hypothesis.

Michele Parry, a student in the Masters of Molecular Medicine Pre-Professional Program at the USF Health Morsani College of Medicine, was working for the former when she experienced the latter. It was an “ah ha!” moment that ended up being a key finding for why certain genes of cancer cells mutate, while others don’t.

Michele Parry.

Parry volunteered in the lab of George Blanck, PhD, professor of molecular medicine, who was studying how the size of a gene’s protein coding region affects it’s the likelihood of becoming mutated. While combing over screen after screen of data – spreadsheets, graphs, and countless lists – she spotted a trend: larger genes are more frequently mutated than smaller ones, and in particular genes encoding cytoskeletal proteins.

“She spotted something that I didn’t and, thanks to that, we were able to run with it,” said Dr. Blanck, whose work looks into the nuances of genes and who pushes to fill the pipeline with talented biomedical sciences students.

The gene mutation work warranted publication, for which Parry was first author. It’s unusual for master’s students to be first author of published research, but Parry’s story is a good example of the experiences students in the USF master’s program can have, Dr. Blanck said.

“This is what master’s students in our program can do,” Dr. Blanck said.  “The role of the student in research is becoming more apparent. Nurturing that experience for a student researcher is directly connected to our mission of teaching.”

Titled “Big genes are big mutagen targets: A connection to cancerous, spherical cells?” in the September 2014 edition of Cancer Letters – the publication resulted in funding for new research looking into how the shape of cancer cells (round versus flat) affects drug resistance.

Dr. Blanck and Wade Sexton, MD, associate professor in the USF Department of Oncologic Sciences and a bladder cancer specialist at Moffitt Cancer Center, were awarded the Anna Valentine Award by Moffitt Cancer Center for new work titled “Cytoskeletal protein related coding region mutations in bladder cancer.”

“Cancers cell have unique characteristics and their shape may affect whether or not they are resistant to drugs,” said Parry.

Parry has a bachelor’s degree in biology and wants to be a physician. Specifically, she wants to be an oncologist. She’s driven to understand the difficult science and realizes she’s lucky to pick it up so fast.

“I’m happy that I’m educated and can understand a lot of this,” she said. “And tutoring the master’s students really helps me cement the molecular biology concepts. We’ll see if I feel the same way as a medical student.”

Parry applied to medical school once and was told to strengthen her resume to increase her likelihood of acceptance.

So, strengthen it she did. Since first applying to medical school in 2012, she has graduated with her master’s degree earning a 4.0 GPA, she now works in Dr. Blanck’s lab and has been published as first author, she is an adjunct professor at St. Petersburg College, and she is the graduate teaching assistant for the master’s program.

“This was supposed to be my year off,” she joked. “But I needed to do all of this to strengthen my candidacy and to prove I could excel at the graduate level.”

Was that “ah ha!” moment proof of her abilities? Parry describes it more as a chance to contribute to promising cancer research.

“It makes me feel valuable,” she said, “and gives me a sense of gratitude.”

Photos by Eric Younghans, USF Health Office of Communications