Imaging the brains of living mice through ultra-thin bone windows, scientists have demonstrated in vivo that amyloid plaques form and reach their full size over a period of weeks. This study, from Jin-Moo Lee and colleagues at Washington University School of Medicine, St. Louis, Missouri, adds a new, partly contradictory perspective to a recent high-profile paper on the dynamics of plaque formation. Lee’s team also provides evidence that plaque growth can be slowed by local gliosis and by small, timely reductions of soluble extracellular Aβ.
The question of how plaques form and grow is one that only a handful of labs are equipped to tackle head-on—that is, using in vivo multiphoton microscopy to watch the process unfold in real time in living animals. Brad Hyman, Brian Bacskai, and others at Massachusetts General Hospital, Charlestown, applied this technique in three mouse models of Alzheimer disease and observed something astonishing: by and large, the plaques they saw were cropping up overnight and hardly growing beyond that (Meyer-Luehmann et al., 2008 and ARF related news story). “I think it was assumed all along (that plaques grow), but when the Hyman paper came out, it was really questioned,” Lee said in an interview with ARF.
Lee and coworkers had been trying to address the same issue with a related approach that is less invasive but more technically demanding than the one used by the MGH group. Both labs used multiphoton microscopy; they differed in how they prepared the cranial windows for visualizing plaques. Hyman and colleagues imaged plaques through a glass slide affixed over a small hole in the mouse’s head. Lee’s team prepared the windows by painstakingly shaving away the skull “to the point where it’s so thin, it’s transparent,” he said. “You’re not opening up the skull, so you’re observing what is presumed to be intact brain without disrupting that integrity.”
To characterize plaque formation and growth, first author Ping Yan and colleagues imaged plaques across several time intervals in six- or 10-month-old APP/PS1 transgenic mice. Plaques in the older mice grew minimally, even over the longest period of observation, i.e., 90 days. However, one-fifth of the plaques in the six-month-old mice at least doubled in size within one week, and over a 28-day period, 62 percent had grown to that extent, some much more. “They saw plaques growing over periods of weeks, whereas we found that they appear within 24 hours,” said Bacskai, a coauthor on the earlier study using the open-skull approach. “We did not see plaques growing the way they do. They saw plaques getting five to six times bigger. We never saw that.”
Lee believes the different methods of preparing the cranial window may account for the apparent discrepancy. Though the thinned-skull method can cause damage to underlying brain tissue if not done carefully, his team chose to proceed with this approach after seeing extensive microglial and astrocytic activation in open-window preparations they had analyzed as a comparison. Other studies have also shown increased gliosis under open-skull but not thinned-skull cranial windows (see, e.g., Holtmaat et al., 2009 and Xu et al., 2007).
In contrast to the substantial plaque growth observed beneath the thin windows, Lee’s team saw very little growth through the open cranial windows they had prepared, reproducing what Hyman’s team had reported using the latter method (Meyer-Luehmann et al., 2008). “The plaques that pop up tend to be small, and they stay small,” Lee said.
In a separate set of experiments, Lee and colleagues treated six-month-old APP/PS1 mice with a γ-secretase inhibitor to determine whether reduced Aβ production affects plaque growth. Many in the field have assumed that plaques grow more slowly when the supply of soluble Aβ in the extracellular space dwindles, but few have attempted to observe directly whether this is true. Lee’s team did so by performing in-vivo microdialysis experiments in collaboration with coauthor David Holtzman, also at Washington University, to measure Aβ levels in the interstitial fluid (ISF) before and after treating mice with the γ-secretase inhibitor.
“If you decrease ISF Aβ by just a little bit, 25 percent over 24 hours, you can actually arrest plaque growth,” Lee said. These findings show “that γ-secretase modulators, or low-dose γ-secretase inhibitors, might be enough to lead to beneficial effects in patients,” Bacskai said. The researchers saw no such effect in 10-month-old mice, though, bolstering a growing view that treatment with Aβ-reducing compounds should begin before extensive plaque formation.
Bacskai remains at odds with the different conclusions on dynamics of plaque growth from the two studies. “Thin skulls are much harder to do, and limit what you can accomplish. You don't get to see as much of the brain,” Bacskai told ARF, noting that open-skull windows offer, in comparison, “a lot of real estate.” In addition, the open-skull approach allows repeat imaging of the same plaques over and over again, whereas with thin-skull windows, researchers can look at the same spot at most two or three times.
The open-skull method has drawbacks, too. “You’re taking out a part of the skull,” Lee said. “In effect, you’re introducing a foreign body—the glass coverslip. It's pretty invasive.”
Bacskai agreed that each technique has pros and cons. Even careful open-skull preparations may show minor damage at the edge of the window, he said, “but we always looked near the center.” Furthermore, switching from one method to another “is not like changing the pH of a buffer. It’s a surgical technique that you need to get good at, and it’s experience-dependent,” he said. “We don’t see this kind of gliosis at all if [the open-skull windows] are done correctly.” A recent study comparing the two methods (Holtmaat et al., 2009) suggested that the increased gliosis in open-skull windows did not seem to make a difference—at least for imaging synaptic spines. Bacskai and members of the Hyman lab have pioneered these in-vivo microscopy techniques for AD research, and Bacskai won a neuroimaging award for a separate study using these techniques at the International Conference on Alzheimer's Disease (ICAD) held last July in Vienna, Austria.
Nevertheless, the new data have made some scientists believe that choice of technique does matter. “This is excellent work,” Bart De Strooper, K.U. Leuven, Belgium, wrote in an e-mail to ARF. “The authors make elegantly the case that the procedures used to visualize amyloid plaques in vivo may strongly affect the generation and dynamics of the plaques.” Based on the new data, Gunnar Gouras, Weill Medical College of Cornell University, New York, suggested that the techniques could have distinct effects on nearby neurons. The findings “provide further evidence for the importance of inflammatory cells in modulating plaque pathology,” he wrote. “One wonders whether the different methods have a differential effect on neuritic dystrophy.” (See full comments below.)
Technical differences aside, Samir Kumar-Singh of the University of Antwerp, Belgium, thinks the new study’s conclusions about how long plaques take to grow “make intuitively more sense.” However, it “doesn’t really matter if it’s one day, two days, 10 days, or 15 days,” he said in a phone interview. “The idea is that plaques grow very quickly at the early stages,” and Aβ-reducing treatments should begin early in the pathogenic process. In an e-mail to ARF, Kumar-Singh noted that the dye (methoxy-X04) used in multiphoton microscopy “only binds fibrillar Aβ, not soluble/oligomeric forms of Aβ that most likely provide the initial nidus of plaque formation.” Therefore, he writes, “I don’t believe that we have had the final word on the temporal kinetics and dynamics of plaque formation.” (See full comment below.)
In the meantime, Lee and Bacskai are pursuing the question from a different angle—by determining what makes plaque growth grind to a halt. “Regardless of the exact rate of plaque formation, they have to stop growing at some point, because your whole brain doesn't turn into one gigantic plaque,” Bacskai said. “If you look at AD brain tissue at any stage, average plaque size is the same. Amyloid burden increases because you have an increased number of plaques, not because they’re bigger. So I would propose there’s some active process that makes a plaque stop growing.” Reactive glial cells could be key players, according to new data from Lee’s lab suggesting that activated astrocytes can inhibit plaque growth. Lee presented some of those unpublished findings at ICAD in Vienna.—Esther Landhuis