Circumscribing Laser Cuts Attenuate Seizure Propagation in a Mouse Model of Focal Epilepsy.

In partial onset epilepsy, seizures arise focally in the brain and often propagate. Patients frequently become refractory to medical management, leaving neurosurgery, which can cause neurologic deficits, as a primary treatment. In the cortex, focal seizures spread through horizontal connections in layers II/III, suggesting that severing these connections can block seizures while preserving function. Focal neocortical epilepsy is induced in mice, sub-surface cuts are created surrounding the seizure focus using tightly-focused femtosecond laser pulses, and electrophysiological recordings are acquired at multiple locations for 3-12 months. Cuts reduced seizure frequency in most animals by 87%, and only 5% of remaining seizures propagated to the distant electrodes, compared to 80% in control animals. These cuts produced a modest decrease in cortical blood flow that recovered and left a ≈20-µm wide scar with minimal collateral damage. When placed over the motor cortex, cuts do not cause notable deficits in a skilled reaching task, suggesting they hold promise as a novel neurosurgical approach for intractable focal cortical epilepsy.

Three-photon excited fluorescence microscopy enables imaging of blood flow, neural structure and inflammatory response deep into mouse spinal cord in vivo.

Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 µm into the mouse spinal cord, in vivo. We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 µm of the murine spinal cord, in vivo.

MousePZT: A simple, reliable, low-cost device for vital sign monitoring and respiratory gating in mice under anesthesia.

Small animal studies in biomedical research often require anesthesia to reduce pain or stress experienced by research animals and to minimize motion artifact during imaging or other measurements. Anesthetized animals must be closely monitored for the safety of the animals and to prevent unintended effects of altered physiology on experimental outcomes. Many currently available monitoring devices are expensive, invasive, or interfere with experimental design. Here, we present MousePZT, a low-cost device based on a simple piezoelectric sensor, with a custom circuit and computer software that allows for measurements of both respiratory rate and heart rate in a non-invasive, minimal contact manner. We find the accuracy of the MousePZT device in measuring respiratory and heart rate matches those of commercial systems. Using the widely-used gas isoflurane and injectable ketamine/xylazine combination, we also demonstrate that changes in respiratory rate are more easily detected and can precede changes in heart rate associated with variations in anesthetic depth. Additional circuitry on the device outputs a respiration-locked trigger signal for respiratory-gating of imaging or other data acquisition and has high sensitivity and specificity for detecting respiratory cycles. We provide detailed instruction documents and all necessary microcontroller and computer software, enabling straightforward construction and utilization of this device.

Extracellular vesicles incorporating retrovirus-like capsids for the enhanced packaging and systemic delivery of mRNA into neurons.

The blood-brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5'-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.

Quasi-analytic solution for real-time multi-exposure speckle imaging of tissue perfusion.

Laser speckle contrast imaging (LSCI) is a widefield imaging technique that enables high spatiotemporal resolution measurement of blood flow. Laser coherence, optical aberrations, and static scattering effects restrict LSCI to relative and qualitative measurements. Multi-exposure speckle imaging (MESI) is a quantitative extension of LSCI that accounts for these factors but has been limited to post-acquisition analysis due to long data processing times. Here we propose and test a real-time quasi-analytic solution to fitting MESI data, using both simulated and real-world data from a mouse model of photothrombotic stroke. This rapid estimation of multi-exposure imaging (REMI) enables processing of full-frame MESI images at up to 8 Hz with negligible errors relative to time-intensive least-squares methods. REMI opens the door to real-time, quantitative measures of perfusion change using simple optical systems.

RNA structures and dynamics with Å resolution revealed by x-ray free-electron lasers.

RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free-electron laser sources to reveal the formation and ready identification of angstrom-scale features in structured and unstructured RNAs. Previously unrecognized structural signatures of RNA secondary and tertiary structures are identified through wide-angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base-paired intermediate to assume a triple-helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. This method may help to rapidly characterize and identify structural elements in nucleic acids in both equilibrium and time-resolved experiments.

Bidirectional Regulation of Motor Circuits Using Magnetogenetic Gene Therapy.

Regulating the activity of discrete neuronal populations in living mammals after delivery of modified ion channels can be used to map functional circuits and potentially treat neurological diseases. Here we report a novel suite of magnetogenetic tools, based on a single anti-ferritin nanobody-TRPV1 receptor fusion protein, which regulated neuronal activity in motor circuits when exposed to magnetic fields. AAV-mediated delivery of a cre-dependent nanobody-TRPV1 calcium channel into the striatum of adenosine 2a (A2a) receptor-cre driver mice led to restricted expression within D2 neurons, resulting in motor freezing when placed in a 3T MRI or adjacent to a transcranial magnetic stimulation (TMS) device. Functional imaging and fiber photometry both confirmed focal activation of the target region in response to the magnetic fields. Expression of the same construct in the striatum of wild-type mice along with a second injection of an AAVretro expressing cre into the globus pallidus led to similar circuit specificity and motor responses. Finally, a mutation was generated to gate chloride and inhibit neuronal activity. Expression of this variant in subthalamic nucleus (STN) projection neurons in PitX2-cre parkinsonian mice resulted in reduced local c-fos expression and a corresponding improvement in motor rotational behavior during magnetic field exposure. These data demonstrate that AAV delivery of magnetogenetic constructs can bidirectionally regulate activity of specific neuronal circuits non-invasively in vivo using clinically available devices for both preclinical analysis of circuit effects on behavior and potential human clinical translation.

RNA structures and dynamics with Å resolution revealed by x-ray free electron lasers.

RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free electron laser sources to reveal the formation and ready identification of Å scale features in structured and unstructured RNAs. New structural signatures of RNA secondary and tertiary structures are identified through wide angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base paired intermediate to assume a triple helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. In addition to understanding how RNA triplexes form and thereby function as dynamic signaling elements, this new method can vastly increase the rate of structure determination for these biologically essential, but mostly uncharacterized macromolecules.

Evaluation of a gain-managed nonlinear fiber amplifier for multiphoton microscopy.

Two-photon excited fluorescence microscopy is a widely-employed imaging technique that enables the noninvasive study of biological specimens in three dimensions with sub-micrometer resolution. Here, we report an assessment of a gain-managed nonlinear (GMN) fiber amplifier for multiphoton microscopy. This recently-developed source delivers 58-nJ and 33-fs pulses at 31-MHz repetition rate. We show that the GMN amplifier enables high-quality deep-tissue imaging, and furthermore that the broad spectral bandwidth of the GMN amplifier can be exploited for superior spectral resolution when imaging multiple distinct fluorophores.

A quasi-analytic solution for real-time multi-exposure speckle imaging of tissue perfusion.

Laser speckle contrast imaging (LSCI) is a widefield imaging technique that enables high spatiotemporal resolution measurement of blood flow. Laser coherence, optical aberrations, and static scattering effects restrict LSCI to relative and qualitative measurements. Multi-exposure speckle imaging (MESI) is a quantitative extension of LSCI that accounts for these factors but has been limited to post-acquisition analysis due to long data processing times. Here we propose and test a real-time quasi-analytic solution to fitting MESI data, using both simulated and real-world data from a mouse model of photothrombotic stroke. This rapid estimation of multi-exposure imaging (REMI) enables processing of full-frame MESI images at up to 8 Hz with negligible errors relative to time-intensive least-squares methods. REMI opens the door to real-time, quantitative measures of perfusion change using simple optical systems.

Vascular oxidative stress causes neutrophil arrest in brain capillaries, leading to decreased cerebral blood flow and contributing to memory impairment in a mouse model of Alzheimer’s disease.

INTRODUCTION: In this study, we explore the role of oxidative stress produced by NOX2-containing NADPH oxidase as a molecular mechanism causing capillary stalling and cerebral blood flow deficits in the APP/PS1 mouse model of AD. METHODS: We inhibited NOX2 in APP/PS1 mice by administering a 10 mg/kg dose of the peptide inhibitor gp91-ds-tat i.p., for two weeks. We used in vivo two-photon imaging to measure capillary stalling, penetrating arteriole flow, and vascular inflammation. We also characterized short-term memory function and gene expression changes in cerebral microvessels. RESULTS: We found that after NOX2 inhibition capillary stalling, as well as parenchymal and vascular inflammation, were significantly reduced. In addition, we found a significant increase in penetrating arteriole flow, followed by an improvement in short-term memory, and downregulation of inflammatory gene expression pathways. DISCUSSION: Oxidative stress is a major mechanism leading to microvascular dysfunction in AD, and represents an important therapeutic target.

Cutting Edge: CCR9 Promotes CD8+ T Cell Recruitment to the Brain during Congenital Cytomegalovirus Infection.

CD8+ T lymphocytes infiltrate the brain during congenital CMV infection and promote viral clearance. However, the mechanisms by which CD8+ T cells are recruited to the brain remain unclear. Using a mouse model of congenital CMV, we found a gut-homing chemokine receptor (CCR9) was preferentially expressed in CD8+ T cells localized in the brain postinfection. In the absence of CCR9 or CCL25 (CCR9's ligand) expression, CD8+ T cells failed to migrate to key sites of infection in the brain and protect the host from severe forms of disease. Interestingly, we found that expression of CCR9 on CD8+ T cells was also responsible for spatial temporal positioning of T cells in the brain. Collectively, our data demonstrate that the CMV-infected brain uses a similar mechanism for CD8+ T cell homing as the small intestine.

Ultrasonically actuated neural probes for reduced trauma and inflammation in mouse brain.

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. Two-photon 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.

Hypoxia-induced carbonic anhydrase mediated dorsal horn neuron activation and induction of neuropathic pain.

ABSTRACT: Neuropathic pain, such as that seen in diabetes mellitus, results in part from central sensitisation in the dorsal horn. However, the mechanisms responsible for such sensitisation remain unclear. There is evidence that disturbances in the integrity of the spinal vascular network can be causative factors in the development of neuropathic pain. Here we show that reduced blood flow and vascularity of the dorsal horn leads to the onset of neuropathic pain. Using rodent models (type 1 diabetes and an inducible endothelial-specific vascular endothelial growth factor receptor 2 knockout mouse) that result in degeneration of the endothelium in the dorsal horn, we show that spinal cord vasculopathy results in nociceptive behavioural hypersensitivity. This also results in increased hypoxia in dorsal horn neurons, depicted by increased expression of hypoxia markers such as hypoxia inducible factor 1α, glucose transporter 3, and carbonic anhydrase 7. Furthermore, inducing hypoxia through intrathecal delivery of dimethyloxalylglycine leads to the activation of dorsal horn neurons as well as mechanical and thermal hypersensitivity. This shows that hypoxic signalling induced by reduced vascularity results in increased hypersensitivity and pain. Inhibition of carbonic anhydrase activity, through intraperitoneal injection of acetazolamide, inhibited hypoxia-induced pain behaviours. This investigation demonstrates that induction of a hypoxic microenvironment in the dorsal horn, as occurs in diabetes, is an integral process by which neurons are activated to initiate neuropathic pain states. This leads to the conjecture that reversing hypoxia by improving spinal cord microvascular blood flow could reverse or prevent neuropathic pain.

Femtosecond optical parametric chirped-pulse amplification in birefringent step-index fiber.

We demonstrate an optical parametric chirped-pulse amplifier (OPCPA) that uses birefringence phase matching in a step-index single-mode optical fiber. The OPCPA is pumped with chirped pulses that can be compressed to sub-30-fs duration. The signal (idler) pulses are generated at 905 nm (1270 nm), have 26 nJ (20 nJ) pulse energy, and are compressible to 70 fs duration. The short compressed signal and idler pulse durations are enabled by the broad bandwidth of the pump pulses. Numerical simulations guiding the design are consistent with the experimental results and predict that scaling to higher pulse energies will be possible. Forgoing a photonic crystal fiber for phase-matching offers practical advantages, including allowing energy scaling with mode area.

VEGF signalling causes stalls in brain capillaries and reduces cerebral blood flow in Alzheimer's mice.

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 and exacerbates short-term memory loss in multiple mouse models of Alzheimer's disease. Here, we report that vascular endothelial growth factor (VEGF) signalling 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 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 endothelial nitric oxide synthase protein concentrations, increased occludin levels and decreased the penetration of circulating Evans Blue dye across the blood-brain barrier into the brain parenchyma, suggesting increased blood-brain barrier 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 signalling in APP/PS1 mice contributes to aberrant endothelial nitric oxide synthase /occludin-associated blood-brain barrier permeability, increases the incidence of capillary stalls, and leads to reductions in cerebral blood flow. Reducing leucocyte adhesion by inhibiting luminal VEGF signalling may provide a novel and well-tolerated strategy for improving brain microvascular blood flow in Alzheimer's disease patients.

Synchronously pumped Raman laser for simultaneous degenerate and nondegenerate two-photon microscopy.

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.

Causes and consequences of baseline cerebral blood flow reductions in Alzheimer's disease.

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.

Dynamic capillary stalls in reperfused ischemic penumbra contribute to injury: A hyperacute role for neutrophils in persistent traffic jams.

Ever since the introduction of thrombolysis and the subsequent expansion of endovascular treatments for acute ischemic stroke, it remains to be identified why the actual outcomes are less favorable despite recanalization. Here, by high spatio-temporal resolution imaging of capillary circulation in mice, we introduce the pathological phenomenon of dynamic flow stalls in cerebral capillaries, occurring persistently in salvageable penumbra after reperfusion. These stalls, which are different from permanent cellular plugs of no-reflow, were temporarily and repetitively occurring in the capillary network, impairing the overall circulation like small focal traffic jams. In vivo microscopy in the ischemic penumbra revealed leukocytes traveling slowly through capillary lumen or getting stuck, while red blood cell flow was being disturbed in the neighboring segments under reperfused conditions. Stall dynamics could be modulated, by injection of an anti-Ly6G antibody specifically targeting neutrophils. Decreased number and duration of stalls were associated with improvement in penumbral blood flow within 2-24 h after reperfusion along with increased capillary oxygenation, decreased cellular damage and improved functional outcome. Thereby, dynamic microcirculatory stall phenomenon can be a contributing factor to ongoing penumbral injury and is a potential hyperacute mechanism adding on previous observations of detrimental effects of activated neutrophils in ischemic stroke.

Vascular contributions to cognitive impairment and dementia (VCID): A report from the 2018 National Heart, Lung, and Blood Institute and National Institute of Neurological Disorders and Stroke Workshop.

Vascular contributions to cognitive impairment and dementia (VCID) are characterized by the aging neurovascular unit being confronted with and failing to cope with biological insults due to systemic and cerebral vascular disease, proteinopathy including Alzheimer's biology, metabolic disease, or immune response, resulting in cognitive decline. This report summarizes the discussion and recommendations from a working group convened by the National Heart, Lung, and Blood Institute and the National Institute of Neurological Disorders and Stroke to evaluate the state of the field in VCID research, identify research priorities, and foster collaborations. As discussed in this report, advances in understanding the biological mechanisms of VCID across the wide spectrum of pathologies, chronic systemic comorbidities, and other risk factors may lead to potential prevention and new treatment strategies to decrease the burden of dementia. Better understanding of the social determinants of health that affect risks for both vascular disease and VCID could provide insight into strategies to reduce racial and ethnic disparities in VCID.