06 Jul 2023

It's 7:30 a.m. and Kuopio University Hospital is humming. On the second floor, neurosurgeons are easing into their day with a team discussion of ventriculo-peritoneal shunting, while next door, a woman in her 70s is being readied to go under the knife. In the weeks before, she entered a diagnosis-cum-research protocol. It showed she was a candidate for this therapeutic procedure while also documenting her cognition, imaging her brain in various ways, and banking blood and CSF for longitudinal research (see Part 1 of this series).

The woman is lying on the operating table, unconscious under general anesthesia. A monitor mounted above her left displays her MRI, marked up with measurements to guide her surgeon's work. The scan shows what brought her here: gaping fluid-filled holes where there should be gray and white matter. A grossly enlarged ventricle is smushing her brain. This excessive fluid pressure is what the team—a surgeon, an anesthesiologist, three nurses—is here to fix. Along the way, they will collect samples for science done in Kuopio and internationally.

The surgeon, Paula Walle, draws three incision sites on the woman's skin: one at the skull's Kocher point, behind her hair line, an inch from midline toward her right ear; another behind her ear, and a third on the right side of her belly. Walle shaves the right half of the woman's head and disinfects her skin from the waistline up. Some bacteria can hide at the bottom of hair follicles, beyond the reach of alcohol, hence the surgeon gently turns the woman's head onto her left cheek, and the nurses cover her entire upper body, save for the incision sites, with yellow sheeting that is glued to her skin. She now looks a modern-day mummy with her left hand peeking out.

Meanwhile, Ville Leinonen, the neurosurgeon who leads the hospital's hydrocephalus shunt biopsy program, checks that supplies are at hand for sample collection: tubes filled with artificial CSF for the cortical pyramid to float in on its imminent journey from the patient's brain to the sectioning vibratome (see Part 1 of this series), a Dewar filled with liquid nitrogen to freeze two needle biopsies, ice to cool the ventricular CSF, saline to rinse, and various tubes to hold all the samples that are soon to come.

Today, like every week, these logistics were the work of the hydrocephalus shunt biopsy program's research nurse. Tiina Laaksonen not only manages the frozen brain samples, she is also the patients' main point of contact. They know her best. Before and after their operation, she, with a neurologist, tests their gait and cognition in the outpatient clinic. Laaksonen coordinates their post-op and annual follow-up visits. She maintains the patient registry. As in most clinical research programs that thrive over time, a devoted research nurse stands at its center. "Tiina handles everything. I could not do a thing without her," Leinonen says.

At the operating table, Walle injects lidocaine-adrenaline at the incision sites. Adrenaline reduces bleeding by constricting blood vessels, especially on the highly vascularized skull. It  also adds a second local pain-killing effect to the anesthesia. "If the patient felt any pain while being under, her blood pressure might go up. This injection stabilizes the anesthesia," she explains.

Walle cuts. First, an inch-wide opening behind the ear. This is where a little valve will go, to adjust the outflow of ventricular fluid. Second, an incision through the belly into the abdominal cavity, where the shunt catheter will end. This cut is when the first biopsy comes out—a bit of skin. Leinonen cleans off the blood with rinses and shakes in saline, then drops the skin into cold culture medium, right in the OR. "Just like with bloody CSF, you won't get good results with blood-contaminated skin," he says.

Next out: 3 mm lumps of fat from under the skin and, if the patient has any, intraperitoneal adipose tissue as well. Also, a tiny clipping of muscle from the abdominal wall. In 2013, while conducting a metabolic study (Takalo et al., 2014), Leinonen and Mikko Hiltunen at UEF decided that banking those tissues might be a good idea, and by now the Kuopio tissue bank stores a lot of it. "We thought at the time that the right research questions will emerge," Leinonen said. A decade later, metabolomic and lipidomic ADRD research is growing rapidly, as is its need for well-phenotyped human samples.

Next, Walle grabs a metal tunneling instrument. It is about 2 feet long. With it, she bores a path, i.e., a 4 mm-wide channel, through the patient's connective tissue between skin and facia, pushing all the way from the ear through the neck, over the chest and to the abdomen. While hard to watch for the uninitiated, this is the safest part of the procedure, Leinonen explains. It's done before the skull is opened, to minimize the time the brain is exposed to air.

Now it's time to create a half-inch-wide burr hole above Brodmann area 8. Walle uses a trepanation drill, the kind that stops automatically when it has passed the skull. "You cannot drill into soft tissue with this," Leinonen said. Through the hole, beyond surprisingly thick walls of bone, soft white tissue comes into view. The dura. From it, a little biopsy square comes out, takes a saline shower, and dives into a Dewar.

Looking beneath the dura now, the surgeon realizes that the left half of the burr hole opens onto a sulcus. This means she will work only through the hole's right half for the remainder of the procedure. Neurosurgeons avoid sulci, because these invaginations of cortex have a nest of blood vessels at their bottom, making gyri a far safer point of entry into the ventricle.

Next, the pyramid extraction. With a dura knife, Walle cuts the pia and the soft, sticky cortical layers, aiming just deep enough to catch a tiny tip of white matter at the bottom. Experience guides her here, not technology. She lifts up the piece, ever so gingerly. "That is the critical part. If you crush it, the cells won't fire," Leinonen says. As he receives the pyramid onto plastic foil and slides it into a sterile tube filled with artificial CSF, he sounds satisfied: "It looks rather nice. I hope they get it working in Tarja's lab."

Now the clock is ticking. Pyramid in hand, Leinonen leaves the OR and hurries to a hospital side entrance. There, he passes it on to a junior scientist, who couriers the precious bit to Tarja Malm's laboratory at the A.I. Virtanen Institute for Molecular Sciences a half-mile away. By bicycle. (That day, Malm had brought her daughter's bike to lend to the postdoc because his was broken.)

Why the bike? "We wanted to use a drone, but they are verboten here so as not to interfere with the emergency helicopter pad on the hospital roof. So, we chose the next-fastest way," Leinonen said. (To learn what happens to the pyramid, see Part 3).

Back in the OR, Walle pulls two more pieces of cortex from the same opening into a biopsy syringe and gently expresses them into saline for rinsing. They look like tiny white worms. Within a minute, they go into liquid nitrogen. They are for snRNA-Seq analyses.

Next, Walle slides in a piece of soft silicon tubing that is as wide as the cortical pyramid she has just removed. This tube, essentially, constitutes the shunt. She knows the tube has penetrated the ventricle when CSF comes out the other end. She catches about 20 ml of it in three separate vials, which go on ice.

The sequence of which tissue the surgeon biopsies at which point in the shunt placement procedure is designed to add neither time nor risk for the patient; in other words, samples are taken during each respective step of the surgery procedure. This is standardized.

Next stop for Leinonen: the hospital lab. There he drops off vials of the patient's blood for routine diagnostic tests of protein, cell counts, glucose.

Then he heads downstairs to the tissue bank in the same building. "It's a bit of a secret place," he says. This newly constructed space houses one of Finland's five regional biobanks. The country in 2012 passed a biobanking law, which regulates how samples from outpatient medical care, research studies, and associated registries across the country are to be stored, shared, and the identities of their donors protected. Even as professor of neurosurgery at Kuopio University Hospital and head of its NPH and Early AD program, Leinonen's access is restricted. He has no key, and has never seen the liquid nitrogen tanks there.

To drop off samples from the still-ongoing surgery, Leinonen gets buzzed in and hands off cortex and ventricular CSF to a dedicated neuroscience technician there. Ulla Lehtoaho will centrifuge the ventricular CSF to pellet, cryoprotect, and freeze its cells for research, then she will aliquot and store the supernatant.

Lehtoaho is busy on this Friday. In addition to samples from this morning's iNPH surgery and a second one to be done in the afternoon, she is processing CSF taken at the hospital's neurology outpatient clinic—again for dual diagnostic and research purposes—from people with Alzheimer's, FTD, Parkinson's, and related diseases.

Also rolling into the biobank today: controls. There are never enough controls in CSF research studies. Once again, to aid diagnosis of the patient at hand, and also to build a collection of comparison samples, KUH physicians draw 1 to 3 ml of CSF whenever they need to place an intrathecal catheter anyway to achieve spinal anesthesia. This could be for hip or knee replacements, gynecology, or urology procedures.

"It does not matter what is the indication. All over the world this is the best way of getting control CSF," Leinonen said. Older patients take memory tests to support their phenotypic classification as controls; many have their data in FinnGen to add genotype information to their file.

Led by Kaj Blennow and Henrik Zetterberg, scientists at UGothenberg's Sahlgrenska University Hospital have been collecting CSF in the course of routine neurology care for many years. Now, Kuopio is doing the same thing as part of Finland's broader effort building a CSF biobank. The cell-preservation protocol, an adaptation of commercial “Mr. Frosty” kits, is but the latest addition to this project.

The Kuopio group send aliquots of CSF to Gothenberg so their collaborators there can analyze larger groups for more power. The CSF cells are going to Beth Stevens and Sam Marsh at the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, for research. Leinonen and Hiltunen hope that FinnGen will add CSF collection in its next project phase, too (see Part 5).

Back in the OR, biopsy collection is finished. Nothing more will come out of the patient. Now it's time for the valve and catheter to go in. This is the moment when, to further enhance sterility during the procedure, the surgeon removes the top set of her two gowns and gloves; a nurse helps her put on fresh pairs.

Walle lifts the shunt catheter out of the antibiotic solution in which it has been waiting. She threads this piece of tubing under the woman's skin through the previously formed tunnel from her ear to its outlet in the belly. She connects its proximal end to the lower outlet of the valve by the ear. She connects the much shorter piece of tubing she previously placed in the patient's ventricle to the valve's upper outlet, running this tubing under the skin to protect it from impact.

Walle tests that the shunt is working. "See, it is producing fluid. The ventricular end is properly in place and there is no blockage along the way," she says, holding up the tube's distal end. Then her hand reaches into the patient's abdominal cavity to make sure the catheter can move freely among the bowels. The droplets of brain fluid exiting here get resorbed amid the general moisture of the peritoneal cavity.

While closing the incisions, the surgeon calmly notes that today's all-female OR team has become “quite normal” in Finland. The country supports work-life balance like few others. Walle went on paid leave for a year when her daughter was born. Upon returning to work, she took the four-day-a-week option, to be mom on Mondays and brain surgeon Tuesdays through Fridays. Her daughter's day care cooks her a warm lunch. And it is free.—Gabrielle Strobel

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References

News Citations

1. Brain Tissue From Living People with Amyloid Plaques Can Fire in a Dish 6 Jul 2023
2. Brain Biopsies and FinnGen Form Wellspring for Functional Genomics 7 Jul 2023

Paper Citations

1. Takalo M, Haapasalo A, Martiskainen H, Kurkinen KM, Koivisto H, Miettinen P, Khandelwal VK, Kemppainen S, Kaminska D, Mäkinen P, Leinonen V, Pihlajamäki J, Soininen H, Laakso M, Tanila H, Hiltunen M. High-fat diet increases tau expression in the brain of T2DM and AD mice independently of peripheral metabolic status. J Nutr Biochem. 2014 Jun;25(6):634-41. Epub 2014 Mar 12 PubMed.

Other Citations

1. Part 1

External Citations

1. NPH and Early AD program

Further Reading

No Available Further Reading