Brain and blood extraction for immunostaining, protein, and RNA measurements after long-term two photon imaging in mice

Nancy E. Ruiz-Uribe, Oliver Bracko

Protocol Exchange (2020)

 View Abstract

This protocol describes a method to extract brain tissue and whole blood samples to perform immunostaining, protein extraction for ELISA, western blot, or RNA extraction for qPCR after long-term in vivo imaging. This protocol is in particular useful to process and maintain valuable tissue samples, allowing for a broad spectrum of analysis and techniques without compromising the quality of the samples.

 Full Access

High fat diet worsens Alzheimer’s disease-related behavioral abnormalities and neuropathology in APP/PS1 mice, but not by synergistically decreasing cerebral blood flow

Oliver Bracko, Lindsay K. Vinarcsik, Jean C. Cruz Hernández, Nancy E. Ruiz-Uribe, Mohammad Haft-Javaherian, Kaja Falkenhain, Egle M. Ramanauskaite, Muhammad Ali, Aditi Mohapatra, Madisen A. Swallow, Brendah N. Njiru, Victorine Muse, Pietro E. Michelucci, Nozomi Nishimura & Chris B. Schaffer

Scientific Reports (2020)

 View Abstract

Obesity is linked to increased risk for and severity of Alzheimer’s disease (AD). Cerebral blood flow (CBF) reductions are an early feature of AD and are also linked to obesity. We recently showed that non-flowing capillaries, caused by adhered neutrophils, contribute to CBF reduction in mouse models of AD. Because obesity could exacerbate the vascular inflammation likely underlying this neutrophil adhesion, we tested links between obesity and AD by feeding APP/PS1 mice a high fat diet (Hfd) and evaluating behavioral, physiological, and pathological changes. We found trends toward poorer memory performance in APP/PS1 mice fed a Hfd, impaired social interactions with either APP/PS1 genotype or a Hfd, and synergistic impairment of sensory-motor function in APP/PS1 mice fed a Hfd. The Hfd led to increases in amyloid-beta monomers and plaques in APP/PS1 mice, as well as increased brain inflammation. These results agree with previous reports showing obesity exacerbates AD-related pathology and symptoms in mice. We used a crowd-sourced, citizen science approach to analyze imaging data to determine the impact of the APP/PS1 genotype and a Hfd on capillary stalling and CBF. Surprisingly, we did not see an increase in the number of non-flowing capillaries or a worsening of the CBF deficit in APP/PS1 mice fed a Hfd as compared to controls, suggesting that capillary stalling is not a mechanistic link between a Hfd and increased severity of AD in mice. Reducing capillary stalling by blocking neutrophil adhesion improved CBF and short-term memory function in APP/PS1 mice, even when fed a Hfd.

 Full Access

Intravital Microscopy of the Beating Murine Heart to Understand Cardiac Leukocyte Dynamics

Nathaniel H. Allan-Rahill, Michael R. E. Lamont, William M. Chilian, Nozomi Nishimura and David M. Small

Frontiers in Immunology (2020)

 View Abstract

Cardiovascular disease is the leading cause of worldwide mortality. Intravital microscopy has provided unprecedented insight into leukocyte biology by enabling the visualization of dynamic responses within living organ systems at the cell-scale. The heart presents a uniquely dynamic microenvironment driven by periodic, synchronous electrical conduction leading to rhythmic contractions of cardiomyocytes, and phasic coronary blood flow. In addition to functions shared throughout the body, immune cells have specific functions in the heart including tissue-resident macrophage-facilitated electrical conduction and rapid monocyte infiltration upon injury. Leukocyte responses to cardiac pathologies, including myocardial infarction and heart failure, have been well-studied using standard techniques, however, certain questions related to spatiotemporal relationships remain unanswered. Intravital imaging techniques could greatly benefit our understanding of the complexities of in vivo leukocyte behavior within cardiac tissue, but these techniques have been challenging to apply. Different approaches have been developed including high frame rate imaging of the beating heart, explantation models, micro-endoscopy, and mechanical stabilization coupled with various acquisition schemes to overcome challenges specific to the heart. The field of cardiac science has only begun to benefit from intravital microscopy techniques. The current focused review presents an overview of leukocyte responses in the heart, recent developments in intravital microscopy for the murine heart, and a discussion of future developments and applications for cardiovascular immunology.

 Full Access

Label-free assessment of hemodynamics in individual cortical brain vessels using third harmonic generation microscopy

Sung Ji Ahn, Nancy E. Ruiz-Uribe, Baoqiang Li, Jason Porter, Sava Sakadzic, and Chris B. Schaffer

Biomedical Optics Express (2020)

 View Abstract

We show that third harmonic generation (THG) microscopy using a 1-MHz train of 1,300-nm femtosecond duration laser pulses enabled visualization of the structure and quantification of flow speed in the cortical microvascular network of mice to a depth of > 1 mm. Simultaneous three-photon imaging of an intravascular fluorescent tracer enabled us to quantify the cell free layer thickness. Using the label-free imaging capability of THG, we measured flow speed in different types of vessels with and without the presence of an intravascular tracer conjugated to a high molecular weight dextran (2 MDa FITC-dextran, 5% w/v in saline, 100 µl). We found a ∼20% decrease in flow speeds in arterioles and venules due to the dextran-conjugated FITC, which we confirmed with Doppler optical coherence tomography. Capillary flow speeds did not change, although we saw a ∼7% decrease in red blood cell flux with dextran-conjugated FITC injection.

 Full Access

Sort by Archive Year

Sort by Principal Investigators