In a narrow medical school hallway, Matt Stewart opened a large cabinet to reveal dozens of shelves stacked with wooden boxes and trays, some at least 100 years old.

Stewart, tall and silver-haired, pulled out one of the trays and showed off its contents: Thin slices of human skull bones and the organs of hearing and balance they contain, stained shades of pink. Affixed to microscope slides, the anatomical bits resembled abstract rubber stamp art, no bigger than thumbprints. “Our Johns Hopkins history,” he said, referring to the university’s collection of specimens from more than 5,000 patients.

Stewart’s research team at Johns Hopkins University in Baltimore had a long, complicated journey to make slides like these in 2021. The researchers need these specimens, sliced from the portion of skull that houses the inner ear, to ask a fundamental question about the novel coronavirus, SARS-CoV-2: Does it directly invade the cells of tissues that enable hearing and balance?

Data on ear problems as they relate to COVID-19, the disease caused by SARS-CoV-2, is spotty. To date, case reports and small studies have found that some COVID-19 patients experience significant and rapid hearing loss, ringing in the ears called tinnitus, or balance issues. Estimates vary on the prevalence of these symptoms, but because the coronavirus has infected hundreds of millions of people, even a few percent of COVID patients experiencing hearing loss would add up to a large increase globally. Yet no causal link has been drawn between the novel coronavirus and auditory symptoms.Hearing problems aren’t even on lists of COVID-19 symptoms, short or long-term, published by the Centers for Disease Control and Prevention.

There are several possible explanations for why the disease might be associated with severe hearing problems, and scientists may never pinpoint all of the underlying mechanisms. But researchers like Stewart are pursuing the theory that the virus could be directly damaging inner ear cells. The coronavirus is already known to infect the cells of the upper nasal cavity, leading to loss of smell. A similar process might occur in the ear, explained Stewart, an associate chief medical officer who specializes in inner ear surgery at the Johns Hopkins Hospital.

The implications would extend beyond the novel coronavirus. Each year some 90,000 people in the United States—that is, 27 out of 100,000—experience sudden hearing loss from damage to the inner ear, and viruses are thought to cause many of these cases. Viruses can also lead to other issues with hearing and balance. But investigating why viruses cause these problems has long been a challenge for scientists. To study these delicate parts, researchers can’t cut up a living person’s inner ear — the research would require the removal of sensitive tissue, risking an injury that might result in total deafness or loss of balance.

The COVID-19 pandemic has motivated researchers to develop new approaches to tackling this longstanding question. At Johns Hopkins, Stewart and colleagues are using cadavers, dissecting the ears using surgical methods from the 1800s, and more recently, with a $7,000 diamond-bladed saw. Meanwhile, a separate group at the Massachusetts Institute of Technology and the Massachusetts Eye and Ear Infirmary has tackled the question by studying human tissue remnants from rare surgeries and by growing inner ear tissue from stem cells—a unique type of cell that can replicate and generate organs. The team published early results in the Nature journal Communications Medicine in October.

Viruses such as SARS-CoV-2, herpes, and the common cytomegalovirus “all have these tentacles that seem to touch the ear, but nobody’s been able to study them because the ear is so inaccessible,” said virologist Lee Gehrke, a senior author of the Communications Medicine study and a professor at both MIT and Harvard University. “So that’s the part that I think I get most excited about,” he said. “Now we have a way to look at these things in a way that we were not able to do before.”

Cadaver can be hard to come by because they require donors. But for Stewart’s team, getting cadavers of patients who had died with COVID-19 wasn’t the most difficult part—Johns Hopkins was initially able to provide three. The bigger challenge was adhering to CDC guidelines. Early in the pandemic, when the research began, no one knew exactly how long the coronavirus could survive under different conditions. The CDC discouraged the use of powered surgical tools like drills, which would be the most obvious choice to get into a cadaver’s ear, but also could shoot viral particles into the air and pose a risk to anyone in the room. Since modern tools were out, Stewart had to rely on surgical techniques from the late 1800s, performed using hand tools that wouldn’t electrically spin up viral particles at high speed.

In an autopsy room, the researchers donned N95 masks and other surgical gear. On each cadaver, they began by making an incision behind the ear, and then found the triangular opening to the mastoid, a part of the skull that “kind of looks like a beehive with a bunch of air cells and very, very, very thin bone,” Stewart said. They operated with a tiny chisel-like instrument called an osteotome in addition to a set of instruments called curettes, “which look like little sharp ice cream scoops,” said Stewart. The curettes can scrape a fraction of a millimeter at a time. From the mastoid they created a 2-millimeter opening—roughly the width of a spaghetti noodle—into the middle ear. They then swabbed inside with a tiny disposable brush.

The Hopkins researchers eventually found the genetic signature of the SARS-CoV-2 coronavirus in two of the three cadavers, confirming that the virus can make its way to the middle ear and mastoid. Stewart and colleagues published these findings in a research letter in the journal JAMA Otolaryngology-Head & Neck Surgery in July 2020, recommending that health care providers wear eye protection and N95 masks during procedures involving the middle ear. (In unpublished follow-up research, the viral signature was found in 60 percent of more than 20 cadavers.)

In an email to Undark, Jameel Muzaffar, a surgeon and researcher at the University of Cambridge and Oto Health in the U.K., said he was not surprised by the signature of SARS-CoV-2 in the middle ear and mastoid since both structures are linked to the nose, where the coronavirus is known to concentrate. Indeed, Stewart said, the virus could have traveled with “infected snot” from elsewhere in the sinuses without necessarily invading precious inner ear cells.

The study does lend support to “the idea that as the virus is present in the middle ear, it could more easily access the inner ear,” potentially causing sudden hearing loss, Muzaffar said. Still, it did not answer the question: Could the coronavirus directly invade and harm the cells of hearing?

A December 2020 study in Laryngoscope explored this question in an organism easier to examine than humans: mice. The novel coronavirus is known to enter cells by interacting with a receptor called ACE-2, which sits on the surface of some human cells. Researchers at the University of Tokyo looked to see if ACE-2 receptors and related proteins are present in mouse ear structures. It turns out, they are.

That study “lit a creeping fire under me,” Stewart said. If his team replicated this in humans, they would need to slice up the inner ear into individual thin cross-sections to analyze under a microscope. But from the time researchers obtain ear samples from cadavers, it can take a year for the bones to soften enough to be sliced thin. That’s a long time to wait in a pandemic.

To look at human cells, Stewart’s team would need to make new slides with thin cross-sections of the organs of hearing and balance like the historical ones in the medical school hallway cabinets. In the previous cadaver study, his team did not have a way to access the inner ear. This time, they’d have to carve their way to that delicate area, and make very fine cuts.

Stewart thought back to when he studied geochemistry as a graduate student. Back then, he had used specialized tools to cut into rocks and gems. “So I had the idea to use a diamond mineralogic saw,” Stewart said. Since the thickness of the blade is just .03 inches, less material would be lost when cutting into temporal bone—a part of the skull encasing the inner ear and balance organs. From there, Stewart reasoned, the research team could rapidly decalcify the cut bones with acid to soften them, so that they could reach those sensitive hearing and balance organs, slice them thin, and perform experiments to look for key cell receptors.

The diamond-bladed saw cost about $7,000—an unusual purchase for an ear surgeon with a wild idea. While the National Institutes of Health had provided funding for Stewart’s previous cadaver study, the agency declined to support this unproven technique, so Stewart directly approached a family that had donated to the hospital, asking if they would support the use of their funds for this project. They did.

Using this technique, Stewart and colleagues successfully created the specimens they needed just about two weeks after the initial surgery. When he got back the first slide last year in early June, “I just sat back in my office and I felt like I had really accomplished something,” Stewart said. His team had surmounted a key obstacle in looking for receptors that the COVID-causing virus could attack.

Preliminary results suggest that they’re on the right track. Using temporal bones from six cadavers that did not have a COVID-19 infection, the researchers found these vulnerable cell receptor types in the middle ear, cochlea, and balance system. That means the novel coronavirus could potentially cause hearing damage by directly invading cells. “That was a big piece of the puzzle,” Stewart said. This has not yet been peer-reviewed, but Stewart presented the results at the American Neurotology Society’s meeting in September 2021, and his team is preparing a manuscript to submit to a journal.

Separately, before the pandemic, a team in the Boston area led by surgeon Konstantina Stankovic, now at Stanford University, was growing inner ear tissues using a type of stem cell that aggregates to form clusters called organoids. But it wasn’t until the advent of COVID that they, in collaboration with researchers at MIT, started using these cells to better understand vulnerability to viruses.

Under a microscope, the organoid tissues resemble what’s inside a human ear, said Gehrke, the virologist, who collaborated with Stankovic. The team has even grown hair cells, sensory receptors that detect movement and enable hearing with tiny stalks called stereocilia sticking out of their surface. These scientists also studied human inner ear tissue from two live patients. It came from a rare surgery that alleviates debilitating vertigo but reduces hearing.

Cells from both the organoids and the patients’ inner ears contained the same proteins that Stewart found in his cadaver research. Gehrke and colleagues then went one step further: They exposed both the organoid and the patient tissues to SARS-CoV-2. As they predicted, the novel coronavirus infected some of the cells. “Their work is so important,” Stewart wrote in a recent email. Gehrke characterized Stewart’s preliminary findings as great news. “Any data that are complementary,” he said, are “very useful.”

Next, Stewart’s team plans to look for evidence of direct invasion in the cells of hearing and balance system organs using samples from COVID-19-positive cadavers. In that way, they could look for possible interactions between human tissue and a virus that had infected it naturally, rather than through an experiment. Stankovic and Gehrke’s team, meanwhile, would like to test experimental treatments in their organoids, Gehrke said, as well as look at other viruses. Both groups want to adapt their models to explore other possible causes of hearing loss from viruses, such as inflammation and immune responses.

All of this may one day lead to better treatments and supportive care for people who struggle with hearing and balance impairment, Stewart said. The subject is personal for him, as he witnessed his father develop hearing loss after years of using dynamite as a field geologist. Because of communication difficulties when sounds are muffled, Stewart added, a patient’s “world kind of contracts.”

With a greater understanding of how viruses interact with the ear, doctors will be able to better help both patients with current COVID disease as well those who have “post-viral effects,” Stewart said.