The process of preserving H. M.'s brain is illustrated on the following pages of this article.
This article is a sample from DISCOVER's special Brain issue, available only on newsstands through June 28.
There is an art to removing the brain from a human cadaver. The donor should be lying faceup, and you should stand just behind the crown of the head. Carefully cut through the skin to expose the skull. Using a neurosurgery drill with a guard plate, cut the bone all the way around the head, above the ears. (It might help to pretend you are a barber giving a monk his tonsure.) This process, called fenestration, is more precise than using a saw. Out of respect for the donor, you do not want to damage the brain.
Remove the top of the skull. With a small scalpel, carefully detach the cranial nerves, which emerge from the brain and thread their way through the skull to the face. As you gently lift the brain away from the skull with your left hand, cut the spinal cord with your right, releasing the brain from the skull. Once the organ is loose in your hand, you must be exceedingly gentle: At this stage it has the consistency of a ripe peach. Weigh it, then treat it with fixatives to preserve the tissue.
Such is the art practiced by Jacopo Annese, a neuroanatomist at the University of California at San Diego. Annese is one of the world’s few experts in dissecting and slicing entire human brains; he has been practicing this craft since 1994. His dream is to create the world’s most complete open-access neuroanatomy library, featuring high-resolution digital images of whole human brain slices. Because of his expertise and this ambition, Annese was chosen by a group of researchers to cut, archive, and curate the most famous brain in neuroscience, that of Henry Molaison—better known to students and researchers worldwide as the legendary amnesiac patient “H. M.”
By the time he was in his twenties, Molaison was incapacitated by epilepsy. His seizures were so frequent and powerful that he could not work. A surgeon named William Beecher Scoville had done experimental surgeries suggesting that severe epilepsy could be treated by removing the parts of the brain that caused the electrical malfunction. He operated on Molaison in 1953, using a suction device to excise a three-inch segment of the medial temporal lobe on both sides. In doing so, Scoville removed most of his patient’s hippocampus, a sea horse–shaped structure that was then thought to be unimportant.
The surgery moderated Molaison’s seizures, as intended. But by destroying both sides of the hippocampus, Scoville had unwittingly also destroyed Molaison’s ability to form long-term memories. For the rest of his long life, Molaison was marooned in the early 1950s, unable to recollect anything new that happened to him. His memories up to 19 months before the surgery were intact, as were his intellect and personality. From that point on, though, he lived in the present. If you had a conversation with him, walked out of the room, and returned after lunch, he would have forgotten everything you said. He would not even remember having met you.
This terrible fate made Molaison a celebrity among neuroscientists, who referred to him by his initials to maintain his anonymity. Because H. M.’s damage was surgically precise and his deficit so specific, he was a perfect test case for the emerging science of memory. He was the man who made it clear that memories were stored in a specific locale, not distributed throughout the brain. H. M. also revealed the difference between skill memories, like how to ride a bicycle, and memories of events; even though he could not retain new information, he could and did get better at tasks. For 55 years he was studied exhaustively by researchers trying to understand how the brain forms and stores its record of the past. By the time of his death, H. M. had been tested by roughly 100 neuroscientists and mentioned in thousands of scientific papers.
Suzanne Corkin, an MIT neuroscientist who worked with H. M. for decades, realized that because his mind had been so thoroughly studied, his brain had unique value. Other distinctive brains—most notably Einstein’s—had been chopped into fragments or lost before they could be studied rigorously. With permission from Molaison’s family, Corkin arranged for H. M.’s brain to be archived and studied. She and other neuroscientists nominated Annese, who had the expertise and the passion to carry out the work. In an ordinary autopsy, just a few chunks would be cut from the brain, but Annese was building a Brain Observatory at U.C. San Diego that would allow the whole organ to be processed and then digitally imaged. Although he alone would do the slicing, Annese wanted to proceed in an open way. Before he began, he got input from researchers around the world. Ultimately, he planned to make H. M.’s digitized brain accessible to all.
Magnetic resonance imaging (MRI) collects low-resolution images, making it possible to see the overall structure of an average brain or get a broad sense of how a disease typically affects neural tissue. It is the workhorse of modern neurology. The old-fashioned craft of tissue sectioning, by contrast, is wholly individual and highly intimate. It reveals the innermost details of one particular brain’s unique folds and neuronal textures. The H. M. project would allow Annese to spend months working with this one brain, probing the microanatomy that made Molaison who he was. “I’m fascinated by spending so much time with one case,” Annese says. “I feel like a watchmaker, doing one very detailed watch.”
As soon as H. M. died, in December 2008, Annese jumped on a red-eye flight to Boston, where after many MRIs, he and neuropathologist Matthew Frosch of Massachusetts General Hospital painstakingly removed Molaison’s brain from his body and fixed it in formaldehyde. Several months later, Annese brought this precious cargo, packed in a cooler and padded with cotton, back to San Diego. With special security permission, he carried the brain onto the plane; it even got its own seat. Once in Annese’s lab, the brain was kept refrigerated under lock and key. A decoy brain labeled “H. M.” was placed in a different refrigerator as an added security measure.
During the spring and summer of 2009, Annese and his collaborators tinkered with the equipment needed to dissect H. M.’s neural tissue into precise slices. The basic technique for brain sectioning was developed by the pathologists of the 19th century, but this project posed unique challenges. Off-the-shelf instruments are designed to handle small chunks of tissue, but whole human brains are huge, by the standards of neuroanatomy. The equipment to prepare, freeze, and slice H. M.’s tissue had to be purpose-built to accommodate an organ of such size. The brain needed to be frozen and sliced at –35 degrees Centigrade, and keeping the temperature constant during slicing was a major concern. A too-warm brain could be gouged by the knife; too cold and it could shatter.
Other essential equipment included Webcams and spotlights. In the interest of open science, Annese planned to Webcast the entire sectioning online.
He and his team expected that just a few brain scientists would tune in, but they were wrong. Annese began slicing on Wednesday, December 2, 2009, exactly one year after H. M.’s death. The next morning, when the researchers returned to the lab, thousands of viewers were waiting for them on the Web. Neuroscientists were watching, but so were all kinds of other people, alerted via blog and Twitter posts. Annese wound up slicing from Thursday morning through late Friday night, inspired by a global audience that eventually totaled about 400,000. On Thursday night, he says, “we were right in the middle of the lesion, so we just went on. We would never be able to forgive ourselves if we left the brain unattended and something went wrong.” By the end, when the brain was fully sliced, Annese was so disoriented that he got lost trying to leave his own lab.
Most of the 2,401 slices of H. M.’s brain are now floating in a cryogenic solution, sequestered in numbered vials inside a locked freezer at –30ºC. Every 36th section is now being stained with Thionine, which highlights fine anatomical structures by dyeing genetic material in each cell a purplish blue. In the coming months, these dyed slices will be placed one by one under a microscope, scanned, and digitized at such high resolution that each 5 × 7 slide will generate a terabyte of visual data. Finally, a computer will reassemble all of that information into a virtual model of H. M.’s brain.
In H. M.’s final gift to science, his brain will yield the first open-access, high-resolution, three-dimensional atlas of the human brain. Something like a Google Earth for the inside of the human head, the atlas will make it possible for neuroscientists (or anyone else) to inspect large features of neuronal architecture, or tiny details down to the level of the cell. Annese hopes this will open the door to new Web-based neuroanatomy initiatives such as collaborative brain mapping to annotate the images. This particular brain, which has been so thoroughly studied in the behavioral domain, may still have some anatomical surprises in store.
In the long term, Annese plans to create a library of hundreds of sliced brains, some from people who have suffered disease or injury and others from individuals with unique skills. Each will be linked to a scientific or anonymized narrative biography of its former owner, an ultimate portrait of one human being. “I want the library to catalog human variability, putting the ‘who’ into neuroscience,” he says. With a large enough collection, it may be possible to draw parallels between brain microstructures and distinct human skills or behaviors. The Brain Observatory team has sectioned about a dozen brains already, and another handful of donors have already agreed to allow Annese to slice up their brains after their death. He also hopes his collection will grow to include brains whose owners possessed an unusual talent—a musician’s, perhaps, or that of someone with synesthesia.
“The idea is that in 5 to 10 years we will have enough cases to look at the microanatomy and solve many questions about human nature, not just about disease,” Annese says. “That would be the ultimate legacy of H. M.”
Continue to page 2 to see the photo gallery that illustrates the process of preserving H. M.'s brain.
This article is a sample from DISCOVER's special Brain issue, available only on newsstands through June 28.
1. The Soak The first step in preparing for the whole-brain slicing takes months: The organ soaks in formaldehyde until it becomes rubbery. Next it is immersed in a mixture of formaldehyde and sucrose. The sugar enters the cells, where it acts like antifreeze, preventing ice crystals from forming when the brain is later frozen to be cut. This brain belonged to a donor who agreed to have his neural tissue archived.
2. The Set After sucrose fully permeates the brain, it can be set in gelatin. Here, H. M.’s brain sits in its purpose-built mold, which will be filled with liquid gelatin and set in a vacuum chamber. The human brain is heavily crenellated, with intricate topography; embedding it in gelatin helps hold all the parts together when it is mounted on glass. According to neuroanatomist Jacopo Annese, “It’s like somebody said: This is a very important old manuscript, and it needs to be preserved. Can you restore this book? Can you tell us what’s in these pages?”
3. The Slice Once the gelatin sets, the brain is frozen, mounted on a motorized tissue-slicing device called a microtome, and sliced. In this picture, H. M.’s brain is being sliced in a plane roughly parallel to the face, in the same orientation as an image from a typical brain scan. As the motorized stage moves backward, a razor-sharp blade peels away a 70-micrometer-thick slice of tissue—like prosciutto at the deli counter, only far thinner. Above: Rubber hoses pipe liquid ethanol at –40°C into cuffs that surround the frozen brain, keeping it precisely chilled. As the blade comes all the way forward, Annese gently lifts the gossamer-thin tissue off the blade with a paintbrush and places it into a well filled with a salt-balanced solution. Next, a digital camera photographs the newly exposed surface. In this picture, Annese is slicing another donor’s brain.
4. The Section Gauzy sections of brain are laid flat on glass slides the size of a postcard. With gentle strokes of a paintbrush, laboratory assistant Natasha Thomas unfolds one section of a donor brain, nudging its complex geometry into proper orientation. Once the brain tissue dries, it can be stained to reveal neuroanatomical structures. Annese holds a whole-brain slice from another amnesic patient. In a healthy brain, the region he peers through would be solid tissue. The gaps were created by a viral infection that damaged the man’s memory. Below: The whole-brain slices in the boxes were treated with a stain that colors cell bodies a deep purplish blue.
5. The Stain This brain section, from another donor, is in the midst of receiving a silver-based stain that darkens myelin, revealing the fibers that connect brain regions. When it is fully “developed,” it will be dark, like the slice Annese holds on the previous page. At the microscopic level, the structure of each brain is unique, reflecting the idiosyncrasies of its former owner. Annese aims to compile an extensive library of sectioned brains whose structures document a wide range of human talents. He already has more donors lined up. “The work we’re doing glorifies you,” Annese says. “It’s like a publisher who decides to publish your biography.”
