Stephen T. Kajdasz

Imaging of amyloid-β deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy

Imaging of amyloid-β deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy

Four-dimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-β ligand in transgenic mice

The lack of a specific biomarker makes preclinical diagnosis of Alzheimers disease (AD) impossible, and it precludes assessment of therapies aimed at preventing or reversing the course of the disease. The development of a tool that enables direct, quantitative detection of the amyloid-β deposits found in the disease would provide an excellent biomarker. This article demonstrates the real-time biodistribution kinetics of an imaging agent in transgenic mouse models of AD. Using multiphoton microscopy, Pittsburgh compound B (PIB) was imaged with sub-μm resolution in the brains of living transgenic mice during peripheral administration. PIB entered the brain quickly and labeled amyloid deposits within minutes. The nonspecific binding was cleared rapidly, whereas specific labeling was prolonged. WT mice showed rapid brain entry and clearance of PIB without any binding. These results demonstrate that the compound PIB has the properties required for a good amyloid-imaging agent in humans with or at risk for AD.

In Vivo Imaging of Amyloid-ß Deposits in Mouse Brain With Multiphoton Microscopy

With the advent of transgenic mouse models expressing cortical amyloid pathology, the potential to study its progression in an intact brain has been realized. Multiphoton microscopy provides a non-destructive means of imaging with micron resolution up to 500 microm deep into the cortex. We detail a surgical procedure and discuss a multiphoton imaging approach that allows for labeling and chronic visualization of amyloid-beta deposits through a cranial window. The ability to monitor these hallmarks of Alzheimers disease enables studies aimed at evaluating the efficacy of treatment and prevention strategies.

Chronic imaging of amyloid plaques in the live mouse brain using multiphoton microscopy

Transgenic mice expressing the human Amyloid Precursor Protein (APP) develop amyloid plaques as they age. These plaques resemble those found in the human disease. Multiphoton laser scanning microscopy combined with a novel surgical approach was used to measure amyloid plaque dynamics chronically in the cortex of living transgenic mice. Thioflavine S (thioS) was used as a fluorescent marker of amyloid deposits. Multiphoton excitation allowed visualization of amyloid plaques up to 200 micrometers deep into the brain. The surgical site could be imaged repeatedly without overt damage to the tissue, and individual plaques within this volume could be reliably identified over periods of several days to several months. On average, plaque sizes remained constant over time, supporting a model of rapid deposition, followed by relative stability. Alternative reporters for in vivo histology include thiazine red, and FITC-labeled amyloid-(Beta) peptide. We also present examples of multi-color imaging using Hoechst dyes and FITC-labeled tomato lectin. These approaches allow us to observe cell nuclei or microglia simultaneously with amyloid-(Beta) deposits in vivo. Chronic imaging of a variety of reporters in these transgenic mice should provide insight into the dynamics of amyloid-(Beta) activity in the brain.

In Vivo Imaging of Alzheimer Pathology in Transgenic Mice using Multiphoton Microscopy

Senile plaques found in Alzheimer’s disease (AD) and in transgenic mouse models of AD are comprised primarily of the amyloid-β peptide. We developed an imaging technique using multiphoton microscopy to enable both detection of amyloid-β pathology in living transgenic mice and functional results of amyloid-β deposition. We used this imaging method to characterize the in vivo kinetics of a novel amyloid-binding agent (6-OH-BTA-1, or Pittsburgh compound B, “PIB”) in transgenic mice. PIB is a novel, thioflavin-T analog that stains plaques, tangles and cerebrovascular amyloid in post-mortem sections of AD brain with high sensitivity and specificity that can cross the blood-brain-barrier. Individual plaques in living transgenic mice could be detected within 1 min after i.v. injection of 2–10 mg/kg PIB. Thirty min after iv injection, the fluorescence in the parenchyma was largely diminished, whereas amyloid-β deposits remained brightly labeled. These findings suggest that PIB is a viable candidate as an in vivo amyloid imaging agent that could allow both diagnostic detection of amyloid pathology and provide a biomarker to evaluate anti-amyloid drug efficacy studies in AD. We also exploited a fluorogenic probe of oxidative stress to detect free radical generation in vivo in these mouse models that is associated specifically with dense-core, but not diffuse, amyloid-β deposits. We then extended the use of multiphoton microscopy and 2’,7’-dichlorodihydrofluorescein (DCF) fluorescence to screen anti-oxidants that may serve a protective role against plaque-derived oxidative stress in an ex vivo system. These results suggest that anti-oxidant therapy may play a beneficial role in the treatment of AD.