Exercise exerts a beneficial effect on the major pathological and clinical symptoms associated with Alzheimer’s disease in humans and mouse models of the disease. While numerous mechanisms for such benefits from exercise have been proposed, a clear understanding of the causal links remains elusive. Recent studies also suggest that cerebral blood flow in the brain of both Alzheimer’s patients and mouse models of the disease is decreased and that the cognitive symptoms can be improved when blood flow is restored. We therefore hypothesized that the mitigating effect of exercise on the development and progression of Alzheimer’s disease may be mediated through an increase in the otherwise reduced brain blood flow. To test this idea, we performed a pilot study to examine the impact of three months of voluntary wheel running in a small cohort of ~1-year-old APP/PS1 mice on short-term memory function, brain inflammation, amyloid deposition, and baseline cerebral blood flow. Our findings that exercise led to a trend toward improved spatial short-term memory, reduced brain inflammation, markedly increased neurogenesis in the dentate gyrus, and a reduction in hippocampal amyloid-beta deposits are consistent with other reports on the impact of exercise on the progression of Alzheimer’s related symptoms in mouse models. Notably, we did not observe any impact of wheel running on overall baseline blood flow nor on the incidence of non-flowing capillaries, a mechanism we recently identified as one contributing factor to cerebral blood flow deficits in mouse models of Alzheimer’s disease. Overall, our findings add to the emerging picture of differential effects of exercise on cognition and blood flow in Alzheimer’s disease pathology by showing that capillary stalling is not decreased following exercise

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.

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.

The insensitivity of multiphoton microscopy to optical scattering enables high-resolution, high-contrast imaging deep into tissue, including in live animals. Scattering does, however, severely limit the use of spectral dispersion techniques to improve spectral resolution. In practice, this limited spectral resolution together with the need for multiple excitation wavelengths to excite different fluorophores limits multiphoton microscopy to imaging a few, spectrally distinct fluorescent labels at a time, restricting the complexity of biological processes that can be studied. Here, we demonstrate a hyperspectral multiphoton microscope that utilizes three different wavelength excitation sources together with multiplexed fluorescence emission detection using angle-tuned bandpass filters. This microscope maintains scattering insensitivity, while providing high enough spectral resolution on the emitted fluorescence and capitalizing on the wavelength-dependent nonlinear excitation of fluorescent dyes to enable clean separation of multiple, spectrally overlapping labels, in vivo. We demonstrated the utility of this instrument for spectral separation of closely overlapped fluorophores in samples containing 10 different colors of fluorescent beads, live cells expressing up to seven different fluorescent protein fusion constructs, and in multiple in vivo preparations in mouse cortex and inflamed skin, with up to eight different cell types or tissue structures distinguished.

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.

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.