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Of Mice and Microscopy – Promising Insights for Alzheimer's Research: July 18, 2007

This image shows a blood vessel affected with cerebrovascular amyloid angiopathy (CAA) in a mouse model of amyloid deposits found in Alzheimer’s disease. Longitudinal imaging of individual segments of CAA-laden blood vessels allows detection of clearance of CAA after treatment with anti-Aβ antibody (Prada et al., J. Neurosci., 2007). Fluorescent angiograms with Texas red dextran were performed to identify fiduciary markers (blue pseudocolor). Vascular and parenchymal Aβ deposits are identified by fluorescence from systemically administered methoxy-XO4 (red pseudocolor). The image was obtained with multiphoton microscopy by Claudia Prada.

Two of the holy grails of Alzheimer’s disease research are early diagnosis and drug therapies to modify the disease outcome. Achieving these goals would dramatically alter the current state of treating this progressive, degenerative disorder that attacks the brain's nerve cells, or neurons, and robs individuals of their memory and ability to reason, communicate, and carry out daily activities. Today, the only way to diagnose the disease definitively is through an autopsy.

What researchers do know is that two types of abnormal lesions clog the brains of individuals with Alzheimer's disease: beta-amyloid plaques – sticky clumps of protein fragments and cellular material that form outside and around neurons; and neurofibrillary tangles – insoluble twisted fibers composed largely of the protein tau that build up inside nerve cells.

To shed light on how the disease begins and the path it takes, researchers at Massachusetts General Hospital in Boston have combined mouse models, an existing optical imaging technology – multiphoton microscopy (MPM) – and unique visualization agents. Their work provides an important step in evaluating Alzheimer’s progression, hastening the discovery of beneficial drugs to treat the disease and change its course.

“We have a limited understanding of the disease’s progression,” says Brian Bacskai, Associate Professor of Neurology at Massachusetts General Hospital in Boston. “Multiphoton microscopy allows us to see what’s happening as the mouse gets sick and to potentially provide therapeutic interventions.”

High Resolution Imaging

The technique has several advantages over other imaging methods. The infrared light used in MPM penetrates deeply into living tissue without damaging it. Traditional approaches use stains that require ultraviolet light that can damage living cells. MPM provides a resolution of 1 µm – 1,000-fold greater than the resolution achieved with positron emission tomography (PET). When combined with special imaging agents, the multiphoton microscope reveals individual blood vessels, neurons, and plaques in the brain. All of this can be done in a living, intact mouse brain but the approach does require optical access to the brain, which means surgically implanting a cranial window in place of a region of skull. (View a video of a 3-dimensional reconstruction of individual senile plaques just below the surface of the brain in a mouse model of Alzheimer's disease.)

To image plaques in humans, researchers use PET. While it is more sensitive in identifying plaques and can image the entire brain, PET does not show individual plaque deposits. “With PET you get an average plaque load for a given cubic millimeter of brain,” says Bacskai, while “MPM shows you exactly what the brain looks like but you can only image a tiny chunk of brain.” Although MPM only images to a depth of approximately 500 µm, amyloid deposits are found in superficial cortex, near the brain’s surface.

These images depict longitudinal imaging of senile plaques during daily treatment with the curry spice component curcumin, which is a natural anti-oxidant and anti-inflammatory agent. The image to the left shows labeled amyloid deposits (red) before treatment, and the image to the right shows the same imaging volume of the brain 7 days after systemic curcumin treatments. Blood vessels are shown in blue (Texas red dextran angiography). Note that individual plaques are cleared or reduced within this time frame. For details, see Garcia-Alloza et al., J. Neurochem., 2007.

Reducing Plague Build-Up

Bacskai’s approach can show “the relationship between the dynamic occurring throughout the anatomy and physiology in the living brain,” explains David Holtzman, Chairman of the Neurology Department at Washington University School of Medicine. He says the technique is particularly useful for studying cerebral amyloid angiopathy (CAA), which consists of the accumulation of amyloid-β (Aβ) peptide in brain blood vessels. Aβ is also the main protein found in amyloid plaques that build up in the brain with Alzheimer’s. In CAA, Aβ builds up in the wall of small arteries in the brain and can cause bleeding.

The researchers found that by administering an antibody continually into the brain over a 2-week period, the CAA was cleared from the blood vessels with no evidence of hemorrhage. This was in contrast to a single dose of antibody administered directly to the brain, which resulted in an initial clearance of CAA but then a significant rate of regrowth of the deposits. By determining the growth pattern of CAA, the group showed that immunotherapy can be tailored to treat both Alzheimer’s and CAA.

Their findings could be significant since the first major clinical trial using an active immunotherapeutic approach with a compound called AN1792 (a synthetic form of the amyloid beta protein) was halted due to brain inflammation in about 6 percent of participants. “When certain antibodies to Aβ interact with CAA, it can cause little and sometimes big hemorrhages,” says Holtzman. “Brian’s work may be a way to get around this problem.”

Testing New Drugs and Imaging Agents

The work being done by Bacskai’s group also provides a springboard for testing a broader range of drugs. “If we can figure out the key steps in the progression of the disease, then we can try to modulate these steps and see if it translates into changes in disease course,” explains Steven Greenberg, Director of Hemorrhagic Stroke Research at Massachusetts General Hospital and a Bacskai collaborator. “This work will shorten the process of identifying a stable of plausible candidate drugs that could modify how the disease progresses.”

In another recent study, Bacskai and his group found that curcumin, a small fluorescent compound in the Indian spice tumeric, can disrupt existing amyloid plaques and partially restore brain structures distorted by the disease in the mouse brain.

Although researchers cannot use MPM to image humans, MPM research can provide critical information about compounds that could be relevant to clinical imaging. In the case of an imaging agent called Pittsburgh compound B (PiB) developed by William Klunk and Chester Mathis at the University of Pittsburgh’s School of Medicine, Bacskai and his team demonstrated in the mouse model that PiB enters the brain quickly, links to amyloid deposits within minutes, and clears rapidly from the brain. The researchers concluded that the finding would translate directly to successful imaging in humans. A 25-center clinical trial is under way to test PiB as a method of labeling amyloid plaques.

A single senile plaque (red) along with neurites expressing yellow fluorescent protein (green) from the living brain of a mouse model of Alzheimer’s disease. Blood vessels are visible with fluorescence angiography following injection with Texas red dextran (blue). The plaque is approximately 30 µm in diameter and is about 150 µm deep from the brain surface. Image acquired by Monica Garcia-Alloza.

A Role in Human Imaging

“Multiphoton microscopy is a tool that can accelerate the development of other tools to image Alzheimer’s disease in humans,” says Bacskai. “To make the leap [to human imaging] we need to develop more or better PET, SPECT, and MRI probes.” MPM enables researchers to study a range of probes, agents that attach to a target substance (in this case, amyloid deposits). They can monitor how the probes cross into the brain and determine what substances or structures the probes attach to. While much work remains, Greenberg is optimistic about MPM’s role in finding new treatments for Alzheimer’s. “Doing a drug trial is costly and cumbersome, like building a luxury liner,” he says. “What makes MPM useful is that it shows the disease process unfolding in front of us. We can evaluate agents that seem to make biological sense but need more evidence to support them before proceeding to a big trial.” Adds Bacskai, “There is a lot more to do.”

This work is supported in part by the National Institute of Biomedical Imaging and Bioengineering and the National Institute on Aging.

References:

Prada CM, Garcia-Alloza M, Betensky RA, Zhang-Nunes SX, Greenberg SM, Bacskai BJ, Frosch MP. Antibody-mediated clearance of amyloid-β peptide from cerebral amyloid angiopathy revealed by quantitative in vivo imaging. J Neurosci 2007;27(8):1973-80.

Garcia-Alloza M, Borrelli LA, Rozkalne A, Hyman BT, Bacskai BJ. Curcumin labels amyloid pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an Alzheimer mouse model. J Neurochem [epub 2007 April 30].

Robbins EM, Betensky RA, Domnitz SB, Purcell SM, Garcia-Alloza M, Greenberg C, Rebeck GW, Hyman BT, Greenberg SM, Frosch MP, Bacskai BJ. Kinetics of cerebral amyloid angiopathy progression in a transgenic mouse model of Alzheimer disease. J Neurosci 2006;26(2):365-71.

Bacskai BJ, Hickey GA, Skoch J, Kajdasz ST, Wang Y, Huang G, Mathis CA, Klunk WE, Hyman BT. Four-dimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-β ligand in transgenic mice. PNAS 2003;100(21):12462-7.

Brian Bacskai