Amyloid-β (Aβ) peptides in cerebrospinal fluid are among the few established molecular signs of impending Alzheimer disease. By contrast, tracking Aβ dynamics directly in the tiny spaces between cells of affected brain areas has proven a far greater challenge. Reporting in the August 29th issue of Science, researchers at Washington University in St. Louis, Missouri, and at the University of Milan and affiliated hospitals in Italy, have done just that in vivo in human brain trauma patients. Using microdialysis to collect brain interstitial fluid (ISF) samples from 18 patients undergoing invasive monitoring for acute brain injury, the scientists found that Aβ levels in brain ISF correlated strongly with neurological status. They dipped as patients declined and rose as they improved. Though somewhat paradoxical, these findings appear to fit with in vitro studies suggesting that neuronal activity boosts Aβ release. The new work also raises intriguing questions about the physiological role of soluble Aβ in the brain, and provides tools to examine how such functions may fade in AD and related conditions.
Earlier in vitro and animal studies (Kamenetz et al., 2003; Cirrito et al., 2005) have linked higher synaptic activity with increased Aβ production, and a recent study has suggested endocytosis as a key mechanism connecting the two (see ARF related news story). Collecting and analyzing hippocampal brain ISF samples from mice in vivo required a fair bit of technical know-how (Cirrito et al., 2003). Tracking ISF Aβ levels in brains of living patients presents additional challenges—not the least of which is the scarcity of individuals willing to have a microdialysis catheter stuck into their skull for study purposes. Acknowledging such concerns, researchers led by David Brody and David Holtzman of Washington University, and Sandra Magnoni of Ospedale Maggiore Policlinico, a major trauma center in Milan, turned to intensive care unit (ICU) patients who were already getting invasive brain procedures done as part of their clinical care. These 18 ICU patients—12 in Milan and six in St. Louis, none diagnosed with AD or dementia—had holes drilled into their heads for pressure monitoring after acute brain injury, and agreed to have a microdialysis catheter placed into subcortical white matter during the same procedure.
Analyzing the microdialysis samples using enzyme-linked immunosorbent assay (ELISA), the researchers measured Aβ levels every 1 to 2 hours in all patients, starting 12 to 48 hours post-injury at the time the catheter was placed. This required some procedural innovation. The standard microdialysis perfusion fluid allows virtually no Aβ recovery, perhaps because Aβ is so sticky, Brody told ARF. In earlier mouse ISF studies, John Cirrito in the Holtzman lab found a way around this by including bovine serum albumin in the perfusion fluid. BSA is a non-specific blocking agent that apparently keeps Aβ from adhering to the collection vials. In the new study, the researchers similarly improved Aβ recovery by spiking the perfusion fluid with sterile human albumin. Brody noted that this modification could impact future studies looking at different Aβ forms, such as oligomers, which might have different binding properties.
Analysis of a small number of trauma patients in an ICU setting allows at best for limited experimentation and control of conditions, and would be expected to produce variable data. Nevertheless, the researchers uncovered a number of surprising trends. First and foremost, the scientists saw an overall rise in brain ISF Aβ levels over the course of the first few days in most patients: median Aβ concentrations at 60 to 72 hours were 59 percent higher than during the first 12 hours of analysis. This trend was no measurement artifact because concentrations of urea, which control for the stability of the microdialysis catheter function, held steady in the ISF samples over the same time frame.
From a scientific perspective, the initially low Aβ in brain ISF was unexpected. Early-life traumatic brain injury has been reported to increase later risk of AD, presumably by setting in motion its pathophysiological manifestations—namely, buildup of toxic Aβ (see ARF Live Discussion). “With that in mind,” Brody said, “we expected a big spike of Aβ immediately after injury that then subsided during recovery.”
They saw the opposite—initially low Aβ levels that steadily rose as the patients recovered. The Aβ measurements seemed to jibe with other wellness parameters routinely used in ICU monitoring. For instance, low brain ISF Aβ was found to correlate with low glucose and a high lactate/pyruvate ratio, indicators of abnormal brain metabolism. Low brain ISF Aβ also correlated with high intracranial pressure and extreme brain temperatures—problems likely to disrupt neuronal function. “Thus, brain ISF Aβ increased as overall physiology normalized,” the authors wrote.
The observed brain Aβ dynamics seemed to make biological sense in light of previous work in mice suggesting that neuronal activity drives Aβ secretion (see ARF related news story). Changes in brain ISF Aβ from baseline appeared to track, and in some cases precede, changes in global neurological status as assessed by Glasgow Coma Scores in 13 patients from whom these serial measurements could be readily obtained. In other words, as patients regained brain function, their Aβ levels increased. Brody noted, however, that electrical activity in the brains of the patients was not directly measured in this study; hence, a connection between improved neurological status and increased synaptic activity can only be inferred indirectly.
The apparent rise in ISF Aβ during the patients’ recovery could reflect not only increased production of Aβ but also a reduction in its elimination, suggested Roy Weller, University of Southampton School of Medicine, United Kingdom, via email (see full comment below). He and colleagues reported recently that levels of soluble Aβ in the brain depend not only on its level of production but also on the efficiency by which it is cleared from ISF (Weller et al., 2008).
An existing research focus on the role for soluble Aβ in AD pathogenesis intensified when researchers led by Dennis Selkoe of Brigham and Women’s Hospital, Boston, published evidence for neurotoxicity of Aβ oligomers isolated from human AD brains (Shankar et al., 2008). Selkoe commented via email that he finds the data by Brody and colleagues “compelling and biologically sensible.”
The results prompt questions about whether Aβ assumes different roles in short-term versus longer-term scenarios, said Doug Smith, director of the Center for Brain Injury and Repair at the University of Pennsylvania School of Medicine in Philadelphia. “In the chronic setting, you might suspect that having a lot of Aβ floating around is not a good thing,” Smith said in a phone interview. “But in the acute setting…you have to consider that there may be something protective there. I’d be curious to know—if these patients go on releasing Aβ in the brain—whether at some point it affects cognition.”
Smith and Brody acknowledge that the new data provide no clear evidence for a pathophysiologic process linking acute brain injury with later development of AD. Along similar lines, Smith and colleagues reported recently that Aβ plaques induced days after traumatic brain injury (TBI) did not seem to result in AD, as long-term TBI survivors had no virtually no Aβ plaques (Chen et al., 2008). Regarding the current study, Brody noted that his team would not have detected an early, transient rise in brain ISF Aβ, as the ISF microdialysis catheters were not placed into the patients’ brains until at least 12 hours post-injury.
Brad Hyman of Massachusetts General Hospital, Charlestown, offered this comment via email: “While the implications for a ‘normal’ function of Aβ are intriguing, it is still not completely clear whether the data reflect an active role for Aβ or simply establish that it is a marker for neuronal activity.”
Sorting out what happens in AD is another key issue for follow-up work, Hyman noted (see full comment below). In brain imaging studies, lower CSF Aβ42 has been correlated with higher levels of brain amyloid, suggesting that amyloid plaques trap Aβ peptides normally destined for export through the CSF (see ARF related news story). Furthermore, in a prospective study by Holtzman and colleagues, cognitively normal people with low concentrations of CSF Aβ42 and Aβ40 (along with high levels of tau, another CSF biomarker) had increased risk of conversion to dementia during a 3-4 year follow-up period (see ARF related news story).
Interestingly, the current study demonstrated that brain ISF and CSF are not as closely linked as many in the field might have hoped. The Aβ dynamics traced in brain ISF—the rise as neurological status improved, the fall as it worsened—was not reflected in ventricular CSF samples taken in the same patients. “If you only looked at CSF, you would have missed those key dynamics,” Brody said. He cautioned, however, that it remains to be seen whether the ISF-CSF mismatch observed in patients with severe brain injuries holds for AD patients, as well. Likewise, because this study looked at ventricular CSF, i.e. sampled in the brain, it is hard to say whether the ISF/CSF differences cast doubt on the validity of lumbar CSF Aβ and tau measurements used as predictive biomarkers in AD, Brody said. (Ventricular CSF was conveniently sampled in the new study because many patients already had ventricular catheters placed for clinical purposes, whereas lumbar punctures can be dangerous in these patients, he said.) “Quite honestly, this study is just the beginning,” acknowledged Brody. “It raises just as many questions as it answers.”—Esther Landhuis