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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.The authors found that white blood cells plug about 2% of capillaries in the brains of Alzheimer’s disease mouse models. When the adhesion of these cells was blocked, cerebral blood flow immediately increased and cognitive performance rapidly improved.

We present a miniature oblique back-illumination microscope (mOBM) for imaging the microcirculation of human oral mucosa, enabling real-time, label-free phase contrast imaging of individual leukocytes circulating in the bloodstream, as well as their rolling and adhesion on vascular walls—the initial steps in leukocyte recruitment that is a hallmark of inflammation. Using the mOBM system, we studied the leukocyte-endothelial interactions in healthy and locally inflamed tissue and observed drastic changes in leukocyte movement (velocity and displacement profile). Our findings suggest that real-time imaging of leukocyte dynamics can provide new diagnostic insights (assessment of inflammation, temporal progression of disease, evaluation of therapeutic response, etc.) that are not available using conventional static parameters such as cell number and morphology.

Here, using two-photon phosphorescence lifetime microscopy, the local oxygen tension in the bone marrow of live mice is found to be quite low, with spatiotemporal variations depending on the blood vessel type, distance to the endosteum, and changes in cellularity after stress. Oxygen tension in live bone marrow Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in many stem cell types. However, local oxygen concentration, for example in the bone marrow, has never been measured directly. Charles Lin and colleagues have now developed a method based on two-photon microscopy to measure the absolute local oxygen tension ( p O 2 ) in the marrow of live animals. Using this method, they found that while vascular density is high throughout the bone marrow, overall oxygenation is quite low and there is heterogeneity in local p O 2 with respect to vessel type and location. For example, surprisingly, the endosteal region is not the region of the lowest p O 2 . After radiation or chemotherapy, bone marrow p O 2 becomes elevated and transplanted haematopoietic stem/progenitor cells do not seek out regions with the lowest p O 2 for homing. Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells 1 . Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types 2 , 3 , 4 . However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis 5 , expression of hypoxia inducible factor-1α ( Hif-1 α) and related genes 6 , and staining with surrogate hypoxic markers (for example, pimonidazole) 6 , 7 , 8 . Here we perform direct in vivo measurements of local oxygen tension ( p O 2 ) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute p O 2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local p O 2 , with the lowest p O 2 (∼9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These p O 2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions 1 , 2 . It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow 3 – 5 . We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches. A dual genetic strategy enables the labelling and in vivo imaging of native long-term haematopoietic stem cells in the mouse calvarial bone marrow.

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols. Bone marrow endothelial cells have dual roles in the regulation of haematopoietic stem cell maintenance and in the trafficking of blood cells between the bone marrow and the blood circulatory system; this study shows that these different functions are regulated by distinct types of endothelial blood vessels with different permeability properties, affecting the metabolic state of their neighbouring stem cells. Bone marrow blood vessel specialization Endothelial cells of the bone marrow modulate both haematopoietic stem cell (HSC) maintenance and the trafficking of blood cells out of the bone marrow. Tsvee Lapidot and colleagues find that these two aspects are controlled by two distinct types of blood vessels in the bone marrow, with different permeability properties and reactive oxygen species (ROS) levels. Less permeable arteries surrounded by pericytes maintain HSCs in a low reactive oxygen species (ROS) state, whereas the more permeable smaller sinusoids promote HSC activation and allow trafficking of immature and mature leukocytes. The authors also show that in conditions that allow for expansion of HSCs, endothelial integrity is increased, with fewer blood cells moving in and out. Disruption of the endothelial barrier has the reverse effects. Elsewhere in this issue ( page 380 ), Anjali Kusumbe et al . demonstrate that Notch signalling in endothelial cells of bone marrow induces change in the capillaries and mesenchymal stem cells of the environment to support HSC amplification.

Cancer cells have the extraordinary evolutionary potential to adapt and acquire resistance to most conventional and targeted therapies. In a new study, Lin et al., develop a systematic approach to identify combination therapies that produce cancer traps, in which evading the first drug makes the cancer vulnerable to the second.

This study investigates the linkage between mesopause gravity waves and equatorial plasma bubbles (EPBs) from 2012 to 2015 using the collocated nightglow measurements, O2 (762 nm) and OI (630 nm), observed by the Visible and near‐Infrared Spectral Imager onboard the International Space Station under the Ionosphere, Mesosphere, upper Atmosphere, and Plasmasphere (ISS‐IMAP/VISI) mission, along with the ionospheric observations from FORMOSAT‐3/COSMIC (F3/C). ISS‐IMAP/VISI observed a persistent three‐peak longitudinal structure of gravity wave activity near the magnetic equator in South American, African, and South Asian sectors across all seasons. The gravity waves also exhibit significant seasonal latitudinal variations, consistent with the seasonal migration of the Inter‐Tropical Convergence Zone (ITCZ). Notably, the longitudinal distribution of gravity wave occurrences aligns well with the longitudinal pattern of EPB occurrence observed by both VISI and F3/C, particularly over the South American‐African sectors, establishing that ITCZ‐driven gravity waves are the potential seeding source for EPBs. Analysis of ionospheric background conditions relevant to Rayleigh‐Taylor instability confirms that while these conditions strongly influence the seasonal‐longitudinal distribution of EPBs, the presence of gravity waves acts as the modulator of EPB occurrence. Overall, the VISI EPB occurrence rates are consistent with the product of VISI gravity wave and favorable background condition probabilities, demonstrating that the climatology of EPBs is synergistically governed by the gravity wave seeding and ionospheric conditions.

Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning.

Some osteoblasts embed within bone matrix, change shape, and become dendrite-bearing osteocytes. The circuitry that drives dendrite formation during “osteocytogenesis” is poorly understood. Here we show that deletion of Sp7 in osteoblasts and osteocytes causes defects in osteocyte dendrites. Profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates defects in osteocyte maturation in the absence of Sp7. Sp7-dependent osteocyte gene networks are associated with human skeletal diseases. Moreover, humans with a SP7 R316C mutation show defective osteocyte morphology. Sp7-dependent genes that mark osteocytes are enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. These findings reveal a role for Sp7 and its target gene Osteocrin in osteocytogenesis, revealing that pathways that control osteocyte development influence human bone diseases. The molecular circuitry that drives dendrite formation during osteocytogenesis remains poorly understood. Here the authors show that deletion of Sp7, a gene linked to rare and common skeletal disease, in mature osteoblasts and osteocytes causes severe defects in osteocyte dendrites.

Purpose[18F]PSMA-1007 offers advantages of low urinary tracer excretion and theoretical improved spatial resolution for imaging prostate cancer. However, non-specific bone lesions (NSBLs), defined as mild to moderate focal bone uptake without a typical morphological correlate on CT, are a common finding on [18F]PSMA-1007 PET/CT. The purpose of this study was to investigate the clinical outcomes of patients with [18F]PSMA-1007 avid NSBLs, to determine whether patients with NSBLs represent a higher risk clinical cohort, and to determine whether SUVmax can be used as a classifier of bone metastasis.MethodsA retrospective audit of 214 men with prostate cancer was performed to investigate the clinical outcomes of [18F]PSMA-1007 avid NSBLs according to defined criteria. We also compared the serum PSA, Gleason score, and uptake time of patients with [18F]PSMA-1007 avid NSBLs to patients without [18F]PSMA-1007 avid bone lesions. Finally, we analysed an SUVmax threshold to identify bone metastases using ROC curve analysis.ResultsNinety-four of 214 patients (43.9%) demonstrated at least one NSBL. No [18F]PSMA-1007 avid NSBLs met criteria for a likely malignant or definitely malignant lesion after a median 15.8-month follow-up interval (11.9% definitely benign, 50.3% likely benign, and 37.7% equivocal). There were no statistically significant differences in serum PSA, Gleason score, and uptake time between patients with [18F]PSMA-1007 avid NSBLs and those without [18F]PSMA-1007 avid bone lesions. All NSBLs with adequate follow-up had SUVmax ≤ 11.1. The value of the highest SUVmax distinguished between NSBLs and definite prostate cancer bone metastases, whereby an SUVmax threshold of ≥ 7.2 maximized the Youden’s index.Conclusion[18F]PSMA-1007 avid NSBLs rarely represent prostate cancer bone metastases. When identified in the absence of definite metastatic disease elsewhere, it is appropriate to classify those with SUVmax < 7.2 as likely benign. NSBLs with SUVmax 7.2–11.1 may be classified as equivocal or metastatic, with patient clinical risk factors, scan appearance, and potential management implications used to guide interpretation.