To understand how amyloid plaques, the troublesome clumps of Aβ, form and grow in the brain, it helps to just watch the action directly. Advances in multiphoton microscopy have made this possible, allowing scientists to see individual plaques develop within the brains of living mice in real time. Researchers in Germany have now refined the technique to track plaque formation and growth in AD mouse models for up to six months—the longest ever recorded. Two new studies show that plaques grow slowly over time and reach a plateau in older, amyloid-laden animals. The new insight into plaque formation dynamics could enable better monitoring of amyloid-lowering compounds in preclinical development.
The first study, led by Mathias Jucker at the University of Tübingen, Germany, appears in this week’s Journal of Neuroscience. The second study, by Jochen Herms, Ludwig-Maximilians University, Munich, and colleagues was posted December 6 online in Acta Neuropathologica.
A few specialized labs have used in vivo multiphoton microscopy to peer into the brains of AD transgenic mice and track plaque growth through tiny cranial windows. Because it is done in live animals, the analysis is painstaking. Scientists typically rely on fluorescent cells and blood vessels as landmarks to realign the field of view for repeated imaging across multiple time points. However, “because the picture is three-dimensional, if your angle is slightly off, your perspective on the plaque changes,” said Jin-Moo Lee of Washington University School of Medicine in St. Louis, Missouri, who has used two-photon imaging to study plaque dynamics in AD mice, but was not involved in the recent studies.
In Jucker’s paper, first authors Jasmin Hefendehl and Bettina Wegenast-Braun, and colleagues describe a new head restraint system that allows more efficient and accurate multiphoton analysis. The researchers drill a 4-mm-wide hole into the mouse’s skull, seal it with a coverslip, and cement onto that a custom-built titanium ring that fits precisely into a small notch on a motorized stage. Instead of using cells and vessels as signposts to find previously imaged brain areas, “you can basically press a button that does that automatically,” Hefendehl told ARF.
Between imaging sessions, the mice scamper freely, and their behavior and lifespan appear unaffected by the titanium structure fixed to their heads. “We have some mice that have lived quite happily with the ring for two years,” Hefendehl said. Mice can be fitted with the rings once their bone structure is calcified—as early as three months of age—she noted.
Using the new method, which will be described in an upcoming issue of Nature Protocols, the researchers watched plaques develop in the brains of APP/PS1 mice starting at three to four months of age, when amyloid pathology is rampant in this strain. Jucker and colleagues imaged over a 25-week period—a record thus far for multiphoton microscopy studies of AD mice. Imaging weekly for the first two months, biweekly for the next month, and monthly up to 25 weeks, the scientists studied nearly 400 plaques in seven mice, each mouse analyzed at six locations. They observed a period where new plaques form rapidly and grow steadily, and a later phase where the number of new plaques forming peters off.
To analyze the dynamics of this process, the scientists first measured the size distribution of plaques in mice of increasing age (four, seven, and nine months). Four-month-old animals had mostly small plaques of less than 16 micrometers across, whereas plaque size shifted toward larger (up to 44 micrometers diameter) in older mice. Consistent with their predominance in younger animals with less advanced disease, new plaques were small—only 13 percent had a diameter over 8 micrometers, whereas more than three quarters of pre-existing plaques were larger than that.
The team also analyzed changes in volume and diameter for new and already formed plaques. In the volume analysis, new plaques showed higher-fold increases over time, whereas existing plaques showed very little change when assessed in this way. However, when growth was measured in terms of diameter, both types of plaque grew at similar rates. This makes mathematical sense, since a linear increase in diameter would mean that small plaques grow more robustly than large ones when comparing fold increases in volume. “The underlying physiological model suggests that new amyloid ‘layers’ are constantly added to the surface of existing plaques—despite the difference in initial size,” the authors write.
Long-term multiphoton imaging shows growth of amyloid plaques (green) in the brains of APP/PS1 transgenic mice starting at 3.7 months of age. Blood vessels are stained red. View larger image. Image credit: Hefendehl et al. The Journal of Neuroscience 2011
In the Acta Neuropathologica paper, first author Steffen Burgold and colleagues used a similar approach to visualize plaques in AD mice. The researchers made a small incision in the skull, glued on a coverslip, then attached a small metal bar containing a hole for a screw, to enable repositioning of the mouse for repeated imaging sessions. Compared to Jucker’s team, they analyzed plaque formation over a shorter period (six weeks), and used an AD strain (Tg2576) with slower amyloidosis. Tg2576 mice start accumulating brain amyloid around nine to 10 months.
In weekly imaging sessions, the researchers studied 83 plaques in 12-month-old Tg2576 mice, and found that newborn plaques were, on average, considerably smaller in volume than pre-existing ones, suggesting that plaques take on the order of weeks to mature. Both types grew steadily over time, though relative volumes increased more quickly for newborn than for pre-existing plaques. These patterns corroborate Jucker’s findings in APP/PS1 mice with amyloid pathology well underway. However, older (18-month-old) Tg2576 mice did not form new plaques, and their pre-existing ones hardly increased in volume, over the six-week imaging period.
Overall, the two studies found that new plaques start off small and steadily bulk up before reaching a maximum girth in older mice with more advanced disease. The observation that plaques stop growing in old mice is not new (see Christie et al., 2001). Why that happens remains speculative. In aging mice with “lots of plaques around, soluble Aβ peptides have many choices as to where they can go, making it harder for the growth of any single plaque to be detected,” Herms suggested. As for the age-related decline in the rate of new plaque formation, Hefendehl proposed that it may be a passive process where “existing plaques take up amyloid-β that is floating around. “If there are no plaques to take up the soluble Aβ, then there could be a critical concentration of Aβ present to seed a new plaque,” she said.
The current data contrast with the results of a multiphoton microscopy study by Brad Hyman and colleagues at Massachusetts General Hospital in Boston. Imaging plaques in three AD strains, including the Tg2576 mice, the scientists found a significant number of plaques that sprang up overnight with no further growth in the subsequent two-week imaging (Meyer-Luehmann et al., 2008 and ARF related news story). “Some plaques were very big. To think they just appeared in 24 hours was quite amazing,” said Herms said of that study.
Lee and colleagues did their own multiphoton analysis that offered a reason for the apparent lack of plaque growth in the Hyman study. The researchers imaged β amyloid in APP/PS1 transgenic mice and found plaques growing steadily over a period of weeks, not days (Yan et al., 2009 and ARF related news story). However, plaque growth was observed only when the scientists used an alternative method that involved paring the skull to near transparency and imaging through those ultra-thin bone windows. When they drilled all the way through to create open-skull windows, like those used in the Jucker and Herms studies, the WashU team saw considerable gliosis, which they believe stunted further growth of plaques that formed at early timepoints. A recent Nature Methods paper describes a new approach that improves the durability of thin-skull windows, and discusses the ongoing controversy around procedure-induced gliosis (Drew et al., 2010).
In their recent study, Herms and colleagues averted potential inflammatory responses associated with the open-skull procedure by waiting three weeks after surgery to begin imaging. Jucker’s group delayed imaging for just one week, but detected no gliosis—or in this case, proliferation of microglia expressing green fluorescent protein. Staining mouse brains for activated microglia (Iba-1) and astrocytes (GFAP) a week after the open-skull procedure also found no differences between surgically treated mice and control animals. In addition, the Jucker lab optimized the surgery so the skull can be lifted carefully without disrupting the dura matter. “By now, up to 90 percent of the craniotomies we do produce no gliosis,” noted Wegenast-Braun.
Regardless of procedural differences, scientists cite imaging duration as the prime reason for the apparent discrepancy between the plaque growth dynamics seen in Hyman’s study versus other reports. In an e-mail to ARF, Jucker noted that he thinks his new data “do not nullify” Hyman’s findings. “It just extends his work by showing that amyloid plaques grow over time if imaging is done over a long period,” Jucker wrote. Melanie Meyer-Luehmann, first author on the Hyman paper, agrees, noting that the two present studies “show very nicely that plaques can indeed appear very quickly and therefore confirm what we previously published in three different APP transgenic lines.” Before joining the Hyman lab as a postdoc, Meyer-Luehmann did her Ph.D. studies under Jucker, and now heads her own research group at Ludwig-Maximilians University in Munich.
Taken together, the current work strengthens an emerging consensus in multiphoton imaging studies—plaques can initially appear quickly, then grow slowly over long periods and taper in aging animals with extensive amyloid pathology. “There are now three independent groups that have confirmed this using different techniques and different animal models,” Lee noted. By helping to flesh out the growth dynamics of new and existing Aβ plaques, the data may improve future preclinical testing of potential amyloid-lowering agents for AD and other diseases.—Esther Landhuis