Despite the key role of the capillaries in neurovascular function, a thorough characterization of cerebral capillary network properties is currently lacking. Here, we define a range of metrics (geometrical, topological, flow, mass transfer, and robustness) for quantification of structural differences between brain areas, organs, species, or patient populations and, in parallel, digitally generate synthetic networks that replicate the key organizational features of anatomical networks (isotropy, connectedness, space-filling nature, convexity of tissue domains, characteristic size). To reach these objectives, we first construct a database of the defined metrics for healthy capillary networks obtained from imaging of mouse and human brains. Results show that anatomical networks are topologically equivalent between the two species and that geometrical metrics only differ in scaling. Based on these results, we then devise a method which employs constrained Voronoi diagrams to generate 3D model synthetic cerebral capillary networks that are locally randomized but homogeneous at the network-scale. With appropriate choice of scaling, these networks have equivalent properties to the anatomical data, demonstrated by comparison of the defined metrics. The ability to synthetically replicate cerebral capillary networks opens a broad range of applications, ranging from systematic computational studies of structure-function relationships in healthy capillary networks to detailed analysis of pathological structural degeneration, or even to the development of templates for fabrication of 3D biomimetic vascular networks embedded in tissue-engineered constructs.

The compromise between lateral resolution and usable imaging depth range is a bottleneck for optical coherence tomography (OCT). Existing solutions for optical coherence microscopy (OCM) suffer from either large data size and long acquisition time or a nonideal point spread function. We present volumetric OCM of mouse brain ex vivo with a large depth coverage by leveraging computational adaptive optics (CAO) to significantly reduce the number of OCM volumes that need to be acquired with a Gaussian beam focused at different depths. We demonstrate volumetric reconstruction of ex-vivo mouse brain with lateral resolution of 2.2  μm, axial resolution of 4.7  μm, and depth range of ∼1.2  mm optical path length, using only 11 OCT data volumes acquired on a spectral-domain OCM system. Compared to focus scanning with step size equal to the Rayleigh length of the beam, this is a factor of 4 fewer datasets required for volumetric imaging. Coregistered two-photon microscopy confirmed that CAO-OCM reconstructions can visualize various tissue microstructures in the brain. Our results also highlight the limitations of CAO in highly scattering media, particularly when attempting to reconstruct far from the focal plane or when imaging deep within the sample.

The health and function of tissue rely on its vasculature network to provide reliable blood perfusion. Volumetric imaging approaches, such as multiphoton microscopy, are able to generate detailed 3D images of blood vessels that could contribute to our understanding of the role of vascular structure in normal physiology and in disease mechanisms. The segmentation of vessels, a core image analysis problem, is a bottleneck that has prevented the systematic comparison of 3D vascular architecture across experimental populations. We explored the use of convolutional neural networks to segment 3D vessels within volumetric in vivo images acquired by multiphoton microscopy. We evaluated different network architectures and machine learning techniques in the context of this segmentation problem. We show that our optimized convolutional neural network architecture with a customized loss function, which we call DeepVess, yielded a segmentation accuracy that was better than state-of-the-art methods, while also being orders of magnitude faster than the manual annotation. To explore the effects of aging and Alzheimer’s disease on capillaries, we applied DeepVess to 3D images of cortical blood vessels in young and old mouse models of Alzheimer’s disease and wild type littermates. We found little difference in the distribution of capillary diameter or tortuosity between these groups, but did note a decrease in the number of longer capillary segments (>75μm) in aged animals as compared to young, in both wild type and Alzheimer’s disease mouse models.

Rapid growth and perivascular invasion are hallmarks of glioblastoma (GBM) that have been attributed to the presence of cancer stem-like cells (CSCs) and their association with the perivascular niche. However, the mechanisms by which the perivascular niche regulates GBM invasion and CSCs remain poorly understood due in part to a lack of relevant model systems. To simulate perivascular niche conditions and analyze consequential changes of GBM growth and invasion, patient-derived GBM spheroids were co-cultured with brain endothelial cells (ECs) in microfabricated collagen gels. Integrating these systems with 3D imaging and biochemical assays revealed that ECs increase GBM invasiveness and growth through interleukin-8 (IL-8)-mediated enrichment of CSCs. Blockade of IL-8 inhibited these effects in GBM-EC co-cultures, while IL-8 supplementation increased CSC-mediated growth and invasion in GBM-monocultures. Experiments in mice confirmed that ECs and IL-8 stimulate intracranial tumor growth and invasion in vivo. Collectively, perivascular niche conditions promote GBM growth and invasion by increasing CSC frequency, and IL-8 may be explored clinically to inhibit these interactions.

Alzheimer’s disease is associated with a 20–30% reduction in cerebral blood flow. In the APP/PS1 mouse model of Alzheimer’s disease, inhibiting neutrophil adhesion using an antibody against the neutrophil specific protein Ly6G was recently shown to drive rapid improvements in cerebral blood flow that was accompanied by an improvement in performance on short-term memory tasks. Here, in a longitudinal aging study, we assessed how far into disease development a single injection of anti-Ly6G treatment can acutely improve short-term memory function. We found that APP/PS1 mice as old as 15–16 months had improved performance on the object replacement and Y-maze tests of spatial and working short-term memory, measured at one day after anti-Ly6G treatment. APP/PS1 mice at 17–18 months of age or older did not show acute improvements in cognitive performance, although we did find that capillary stalls were still reduced and cerebral blood flow was still increased by 17% in 21–22-months-old APP/PS1 mice given anti-Ly6G antibody. These data add to the growing body of evidence suggesting that cerebral blood flow reductions are an important contributing factor to the cognitive dysfunction associated with neurodegenerative disease. Thus, interfering with neutrophil adhesion could be a new therapeutic approach for Alzheimer’s disease.

Vascular dysfunction is correlated to the incidence and severity of Alzheimer’s disease. In a mouse model of Alzheimer’s disease (APP/PS1) using in vivo, time-lapse, multiphoton microscopy, we found that occlusions of the microvasculature alter amyloid-beta (Ab) plaques. We used several models of vascular injury that varied in severity. Femtosecond laser induced occlusions in single capillaries generated a transient increase in small, cell-sized, Ab deposits visualized with methoxy-X04, a label of fibrillar Ab. After occlusions of penetrating arterioles, some plaques changed morphology, while others disappeared, and some new plaques appeared within a week after the lesion. Antibody labeling of Ab revealed a transient increase in non-fibrillar Ab one day after the occlusion that coincided with the disappearance of methoxy-X04- labeled plaques. Four days after the lesion, anti-Ab labeling decreased and only remained in patches unlabeled by methoxy-X04 near microglia. Histology in two additional models, sparse embolic occlusions from intracarotid injections of beads and infarction from photothrombosis, demonstrated increased labeling intensity in plaques after injury. These results suggest that microvascular lesions can alter the deposition and clearance of Ab and confirm that Ab plaques are dynamic structures, complicating the interpretation of plaque burden as a marker of Alzheimer’s disease progression

Cerebral blood flow (CBF) reductions in Alzheimer’s disease patients and related mouse models have been recognized for decades, but the underlying mechanisms and resulting consequences for Alzheimer’s disease pathogenesis remain poorly understood. In APP/PS1 and 5xFAD mice we found that an increased number of cortical capillaries had stalled blood flow as compared to in wild-type animals, largely due to neutrophils that had adhered in capillary segments and blocked blood flow. Administration of antibodies against the neutrophil marker Ly6G reduced the number of stalled capillaries, leading to both an immediate increase in CBF and rapidly improved performance in spatial and working memory tasks. This study identified a previously uncharacterized cellular mechanism that explains the majority of the CBF reduction seen in two mouse models of Alzheimer’s disease and demonstrated that improving CBF rapidly enhanced short-term memory function. Restoring cerebral perfusion by preventing neutrophil adhesion may provide a strategy for improving cognition in Alzheimer’s disease patients

n vivo optical imaging has enabled detailed studies of cellular dynamics in the brain of rodents in both healthy and diseased states. Such studies were made possible by three advances: surgical preparations that give optical access to the brain; strategies for in vivo labeling of cells with structural and functional fluorescent indicators; and optical imaging techniques that are relatively insensitive to light scattering by tissue. In vivo imaging in the rodent spinal cord has lagged behind than that in the brain, largely due to the anatomy around the spinal cord that complicates the surgical preparation, and to the strong optical scattering of the dorsal white matter that limits the ability to image deep into the spinal cord. Here, we review recent advances in surgical methods, labeling strategies, and optical tools that have enabled in vivo, high-resolution imaging of the dynamic behaviors of cells in the spinal cord in mice. Surgical preparations that enable long-term optical access and robust stabilization of the spinal cord are now available. Labeling strategies that have been used in the spinal cord tend to follow those that have been used in the brain, and some recent advances in genetically-encoded labeling strategies remain to be capitalized on. The optical imaging methods used to date, including two photon excited fluorescence microscopy, are largely limited to imaging the superficial layers of the spinal cord by the optical scattering of the white matter. Finally, we show preliminary data that points to the use of higher-order nonlinear optical processes, such as three photon excited fluorescence, as a means to image deeper into the mouse spinal cord.

The ApoE4 allele is associated with increased risk of small vessel disease, which is a cause of vascular cognitive impairment. Here, we report that mice with targeted replacement (TR) of the ApoE gene with human ApoE4 have reduced neocortical cerebral blood flow compared to ApoE3-TR mice, an effect due to reduced vascular density rather than slowing of microvascular red blood cell flow. Furthermore, homeostatic mechanisms matching local delivery of blood flow to brain activity are impaired in ApoE4-TR mice. In a model of cerebral hypoperfusion, these cerebrovascular alterations exacerbate damage to the white matter of the corpus callosum and worsen cognitive dysfunction. Using 3-photon microscopy we found that the increased white matter damage is linked to an enhanced reduction of microvascular flow resulting in local hypoxia. Such alterations may be responsible for the increased susceptibility to hypoxic-ischemic lesions in the subcortical white matter of individuals carrying the ApoE4 allele.

Cerebral microbleeds are linked to cognitive decline, but it remains unclear how they impair neuronal function. Infarction is not typically observed near microbleeds, suggesting more subtle mechanisms, such as inflammation, may play a role. Because of their small size and largely asymptomatic nature, real-time detection and study of spontaneous cerebral microbleeds in humans and animal models are difficult. We used in vivo 2-photon microscopy through a chronic cranial window in adult mice to follow the inflammatory response after a cortical microhemorrhage of ≈100 μm diameter, induced by rupturing a targeted cortical arteriole with a laser. The inflammatory response included the invasion of blood-borne leukocytes, the migration and proliferation of brain-resident microglia, and the activation of astrocytes. Nearly all inflammatory cells responding to the microhemorrhage were brain-resident microglia, but a small number of CX3CR1+ and CCR2+ macrophages, ultimately originating from the invasion of blood-borne monocytes, were also found near the lesion. We found a coordinated pattern of microglia migration and proliferation, where microglia within 200 μm of the microhemorrhage migrated toward the lesion over hours to days. In contrast, microglia proliferation was not observed until ≈40 hours after the lesion and occurred primarily in a shell-shaped region where the migration of microglia decreased their local density. These data suggest that local microglia density changes may trigger proliferation. Astrocytes activated in a similar region as microglia but delayed by a few days. By 2 weeks, this inflammatory response had largely resolved.Although microhemorrhages are small in size, the brain responds to a single bleed with an inflammatory response that involves brain-resident and blood-derived cells, persists for weeks, and may impact the adjacent brain microenvironment.