Background: Minimally-invasive ablation with radio frequency (RF) and cryoablation have been widely adopted to treat conditions with aberrant neural activity such as excessive mucus production in rhinitis, but neurological and inflammatory effects on treated tissues are poorly understood. Objective: To gain an understanding of the physiological changes caused by nerve ablation using RF and cryoablation devices. Methods: Using clinical devices for rhinitis treatment that ablate nerves with access from the nasal cavity, we applied temperature-controlled RF and cryoablation to rat sciatic nerves. To model the ablation through mucosal tissue similarly to the rhinitis procedure, RF ablation and cryoablation were applied through a layer of muscle. Results: Both ablation techniques induced acute and sustained neurodegeneration visualized with histological sections at two days and one month after treatment. After both treatments, rats showed a change in muscle tone, but small increases in sensitivity measured by a von Frey test were only observed 2 days after cryoablation and one month after the RF ablation. Both treatments caused reductions in nerve conduction velocity at one month after treatment. Inflammation in treated nerves and surrounding tissues that persisted to one month. Conclusions: The two neurolytic devices used in the clinic work similarly by axonal disintegration and which leads to disruption of electrical signals. The data suggest that these methods are effective methods of nerve ablation that could be used to treat diseases related to elevated neuron activity such as rhinitis.

Electrical neural recordings measured using direct electrical interfaces with neural tissue suffer from a short lifespan because the signal strength decreases over time. The inflammatory response to the inserted microprobe can create insulating tissue over the electrical interfaces, reducing the recorded signal below noise levels. One of the factors contributing to this inflammatory response is the tissue damage caused during probe insertion. Here, we explore the use of ultrasonic actuation of the neural probe during insertion to minimize tissue damage in mice. Silicon neural microprobes were designed and fabricated with integrated electrical recording sites and piezoelectric transducers. The microprobes were actuated at ultrasonic frequencies using integrated piezoelectric transducers. The microprobes were inserted into mouse brains under a glass window over the brain surface to image the tissue surrounding the probe using two-photon microscopy. The mechanical force required to penetrate the tissue was reduced by a factor of 2–3 when the microprobe was driven at ultrasonic frequencies. Tissue histology at the probe insertion site showed a reduced area of damage and decreased microglia counts with increasing ultrasonic actuation of the probes. Twophoton imaging of the microprobe over weeks demonstrated stabilization of the inflammatory response. Recording of electrical signals from neurons over time suggests that microprobes inserted using ultrasound have a higher signal-to-noise ratio over an extended time period.

Increased incidence of stalled capillary blood flow caused by adhesion of leucocytes to the brain microvascular endothelium leads to a 17% reduction of cerebral blood flow (CBF) and exacerbates short-term memory loss in multiple mouse models of Alzheimer’s disease. Here, we report that Vascular Endothelial Growth Factor (VEGF) signaling at the luminal side of the brain microvasculature plays an integral role in the capillary stalling phenomenon of the APP/PS1 mouse model. Administration of the anti-mouse VEGF-A164 antibody, an isoform that inhibits blood brain barrier (BBB) hyperpermeability, reduced the number of stalled capillaries within an hour of injection, leading to an immediate increase in average capillary blood flow but not capillary diameter. VEGF-A inhibition also reduced the overall eNOS protein concentrations, increased occludin levels, and decreased the penetration of circulating Evans Blue dye across the BBB into the brain parenchyma, suggesting increased BBB integrity. Capillaries prone to neutrophil adhesion after anti-VEGF-A treatment also had lower occludin concentrations than flowing capillaries. Taken together, our findings demonstrate that VEGF-A signaling in APP/PS1 mice contributes to aberrant eNOS/occludin- associated BBB permeability, increases the incidence of capillary stalls, and leads to reductions in CBF. Reducing leucocyte adhesion by inhibiting luminal VEGF signaling may provide a novel and well-tolerated strategy for improving brain microvascular blood flow in Alzheimer’s disease. patients.

Reductions of baseline cerebral blood flow (CBF) of ∼10–20% are a common symptom of Alzheimer’s disease (AD) that appear early in disease progression and correlate with the severity of cognitive impairment. These CBF deficits are replicated in mouse models of AD and recent work shows that increasing baseline CBF can rapidly improve the performance of AD mice on short term memory tasks. Despite the potential role these data suggest for CBF reductions in causing cognitive symptoms and contributing to brain pathology in AD, there remains a poor understanding of the molecular and cellular mechanisms causing them. This review compiles data on CBF reductions and on the correlation of AD-related CBF deficits with disease comorbidities (e.g. cardiovascular and genetic risk factors) and outcomes (e.g. cognitive performance and brain pathology) from studies in both patients and mouse models, and discusses several potential mechanisms proposed to contribute to CBF reductions, based primarily on work in AD mouse models. Future research aimed at improving our understanding of the importance of and interplay between different mechanisms for CBF reduction, as well as at determining the role these mechanisms play in AD patients could guide the development of future therapies that target CBF reductions in AD.

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase-mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoa1AR) and cAMP (optoB2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

Two-photon fluorescence microscopy is a nonlinear imaging modality frequently used in deep-tissue imaging applications. A tunable-wavelength multicolor short-pulse source is usually required to excite fluorophores with a wide range of excitation wavelengths. This need is most typically met by solid-state lasers, which are bulky, expensive, and complicated systems. Here, we demonstrate a compact, robust fiber system that generates naturally synchronized femtosecond pulses at 1050 nm and 1200 nm by using a combination of gain-managed and Raman amplification. We image the brain of a mouse and view the blood vessels, neurons, and other cell-like structures using simultaneous degenerate and nondegenerate excitation.

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.