Whole-body cellular mapping in mouse using standard IgG antibodies.

Whole-body imaging techniques play a vital role in exploring the interplay of physiological systems in maintaining health and driving disease. We introduce wildDISCO, a new approach for whole-body immunolabeling, optical clearing and imaging in mice, circumventing the need for transgenic reporter animals or nanobody labeling and so overcoming existing technical limitations. We identified heptakis(2,6-di-O-methyl)-ß-cyclodextrin as a potent enhancer of cholesterol extraction and membrane permeabilization, enabling deep, homogeneous penetration of standard antibodies without aggregation. WildDISCO facilitates imaging of peripheral nervous systems, lymphatic vessels and immune cells in whole mice at cellular resolution by labeling diverse endogenous proteins. Additionally, we examined rare proliferating cells and the effects of biological perturbations, as demonstrated in germ-free mice. We applied wildDISCO to map tertiary lymphoid structures in the context of breast cancer, considering both primary tumor and metastases throughout the mouse body. An atlas of high-resolution images showcasing mouse nervous, lymphatic and vascular systems is accessible at http://discotechnologies.org/wildDISCO/atlas/index.php .

Structural changes of CA1 pyramidal neurons after stroke in the contralesional hippocampus.

Significant progress has been made with regard to understanding how the adult brain responds after a stroke. However, a large number of patients continue to suffer lifelong disabilities without adequate treatment. In the present study, we have analyzed possible microanatomical alterations in the contralesional hippocampus from the ischemic stroke mouse model tMCAo 12-14 weeks after transient middle cerebral artery occlusion. After individually injecting Lucifer yellow into pyramidal neurons from the CA1 field of the hippocampus, we performed a detailed three-dimensional analysis of the neuronal complexity, dendritic spine density, and morphology. We found that, in both apical (stratum radiatum) and basal (stratum oriens) arbors, CA1 pyramidal neurons in the contralesional hippocampus of tMCAo mice have a significantly higher neuronal complexity, as well as reduced spine density and alterations in spine volume and spine length. Our results show that when the ipsilateral hippocampus is dramatically damaged, the contralesional hippocampus exhibits several statistically significant selective alterations. However, these alterations are not as significant as expected, which may help to explain the recovery of hippocampal function after stroke. Further anatomical and physiological studies are necessary to better understand the modifications in the "intact" contralesional lesioned brain regions, which are probably fundamental to recover functions after stroke.

Focal Cerebral Ischemia Induces Global Subacute Changes in the Number of Neuroblasts and Neurons and the Angiogenic Factor Density in Mice.

Background and Objectives: Dissecting the complex pathological cascade of an ischemic stroke in preclinical models is highly warranted to understand the course of this disease in humans. Neurogenesis and angiogenesis are integral for post-stroke recovery, yet it is not clear how these processes are altered months after an ischemic stroke. In this study, we investigated the changes that take place subacutely after focal cerebral ischemia in experimental adult male mice. Materials and Methods: Male 12-week-old C57BL/6 mice underwent a 60 min long fMCAo or sham surgery. Two months after the procedure, we examined the immunohistochemistry to assess the changes in neuroblast (DCX) and differentiated neuron (NeuN) numbers, as well as the density of the pro-angiogenic factor VEGF. Results: We found decreased neuroblast numbers in both brain hemispheres of the fMCAo mice: by more than 85% in the dentate gyrus and by more than 70% in the subventricular zone. No neuroblasts were found in the contralateral hemisphere of the fMCAO mice or the sham controls, but a small population was detected in the ipsilateral ischemic core of the fMCAo mice. Intriguingly, the number of differentiated neurons in the ipsilateral ischemic core was lower by 20% compared to the contralateral hemisphere. VEGF expression was diminished in both brain hemispheres of the fMCAo mice. Conclusions: Our current report shows that focal cerebral ischemia induces changes in neuroblast numbers and the pro-angiogenic factor VEGF in both cerebral hemispheres 2 months after an fMCAo in mice. Our data show that focal cerebral ischemia induces a long-term regenerative response in both brain hemispheres.

Microanatomical study of pyramidal neurons in the contralesional somatosensory cortex after experimental ischemic stroke.

At present, many studies support the notion that after stroke, remote regions connected to the infarcted area are also affected and may contribute to functional outcome. In the present study, we have analyzed possible microanatomical alterations in pyramidal neurons from the contralesional hemisphere after induced stroke. We performed intracellular injections of Lucifer yellow in pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere in an ischemic stroke mouse model. A detailed 3-dimensional analysis of the neuronal complexity and morphological alterations of dendritic spines was then performed. Our results demonstrate that pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere show selective changes in their dendritic arbors, namely, less dendritic complexity of the apical dendritic arbor-but no changes in the basal dendritic arbor. In addition, we found differences in spine morphology in both apical and basal dendrites comparing the contralesional hemisphere with the lesional hemisphere. Our results show that pyramidal neurons of remote areas connected to the infarct zone exhibit a series of selective changes in neuronal complexity and morphological distribution of dendritic spines, supporting the hypothesis that remote regions connected to the peri-infarcted area are also affected after stroke.

Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

Perfusion pressure determines vascular integrity and histomorphological quality following perfusion fixation of the brain.

INTRODUCTION: Histology on fixed brain tissue is a key technique to investigate the pathophysiology of neurological disorders. Best results are obtained by perfusion fixation, however, multiple protocols are available and so far the optimal perfusion pressure (PP) for the preservation of brain tissue while also maintaining vascular integrity is not defined. Therefore, the aim of our study was to investigate the effect of different PPs on the cerebral vasculature and to define the PP optimal for the preservation of both vascular integrity and tissue fixation. MATERIAL AND METHODS: Male C57Bl6 mice, 8 weeks old, were perfused with PPs of 50/125/300 mmHg (series I) or 50/100/150/300 mmHg (series II). In series I, vascular integrity, e.g. BBB permeability, vessel diameter, and occurrence of vasospasms were investigated by spectrophotometry, light-sheet and 2-photon microscopy, respectively. In series II, we investigated vascular and neuronal artifacts and the occurrence of hemorrhage or microthrombi by light microscopy. RESULTS: While a PP below the physiological systolic blood pressure results in the collapse of parenchymal vessels and formation of microvasospasms and microclots, a PP above the physiological systolic blood pressure dilates cerebral vessels, induces microvasospasms and disrupts the BBB. In terms of tissue integrity, our results confirm that higher PPs lead to fewer artifacts such as dark neurons or perivascular courts. CONCLUSION: Our study demonstrates that the PP critically affects both vascular and tissue integrity in brain tissue preserved by perfusion fixation. A PP between 125 and 150 mmHg is optimal for the preservation of the cerebral vasculature and neuronal structures.

Dynamic tracing using ultra-bright labeling and multi-photon microscopy identifies endothelial uptake of poloxamer 188 coated poly(lactic-co-glycolic acid) nano-carriers in vivo.

The potential of poly(lactic-co-glycolic acid) (PLGA) to design nanoparticles (NPs) and target the central nervous system remains to be exploited. In the current study we designed fluorescent 70-nm PLGA NPs, loaded with bulky fluorophores, thereby making them significantly brighter than quantum dots in single-particle fluorescence measurements. The high brightness of NPs enabled their visualization by intravital real-time 2-photon microscopy. Subsequently, we found that PLGA NPs coated with pluronic F-68 circulated in the blood substantially longer than uncoated NPs and were taken up by cerebro-vascular endothelial cells. Additionally, confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1 h after systemic injection and were more readily taken up by endothelial cells in peripheral organs. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers.

RIPK1 or RIPK3 deletion prevents progressive neuronal cell death and improves memory function after traumatic brain injury.

Traumatic brain injury (TBI) causes acute and subacute tissue damage, but is also associated with chronic inflammation and progressive loss of brain tissue months and years after the initial event. The trigger and the subsequent molecular mechanisms causing chronic brain injury after TBI are not well understood. The aim of the current study was therefore to investigate the hypothesis that necroptosis, a form a programmed cell death mediated by the interaction of Receptor Interacting Protein Kinases (RIPK) 1 and 3, is involved in this process. Neuron-specific RIPK1- or RIPK3-deficient mice and their wild-type littermates were subjected to experimental TBI by controlled cortical impact. Posttraumatic brain damage and functional outcome were assessed longitudinally by repetitive magnetic resonance imaging (MRI) and behavioral tests (beam walk, Barnes maze, and tail suspension), respectively, for up to three months after injury. Thereafter, brains were investigated by immunohistochemistry for the necroptotic marker phosphorylated mixed lineage kinase like protein(pMLKL) and activation of astrocytes and microglia. WT mice showed progressive chronic brain damage in cortex and hippocampus and increased levels of pMLKL after TBI. Chronic brain damage occurred almost exclusively in areas with iron deposits and was significantly reduced in RIPK1- or RIPK3-deficient mice by up to 80%. Neuroprotection was accompanied by a reduction of astrocyte and microglia activation and improved memory function. The data of the current study suggest that progressive chronic brain damage and cognitive decline after TBI depend on the expression of RIPK1/3 in neurons. Hence, inhibition of necroptosis signaling may represent a novel therapeutic target for the prevention of chronic post-traumatic brain damage.

Ultrabright Fluorescent Polymeric Nanoparticles with a Stealth Pluronic Shell for Live Tracking in the Mouse Brain.

Visualizing single organic nanoparticles (NPs) in vivo remains a challenge, which could greatly improve our understanding of the bottlenecks in the field of nanomedicine. To achieve high single-particle fluorescence brightness, we loaded polymer poly(methyl methacrylate)-sulfonate (PMMA-SO3H) NPs with octadecyl rhodamine B together with a bulky hydrophobic counterion (perfluorinated tetraphenylborate) as a fluorophore insulator to prevent aggregation-caused quenching. To create NPs with stealth properties, we used the amphiphilic block copolymers pluronic F-127 and F-68. Fluorescence correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that pluronics remained at the NP surface after dialysis (at one amphiphile per 5.5 nm2) and prevented NPs from nonspecific interactions with serum proteins and surfactants. In primary cultured neurons, pluronics stabilized the NPs, preventing their prompt aggregation and binding to neurons. By increasing dye loading to 20 wt % and optimizing particle size, we obtained 74 nm NPs showing 150-fold higher single-particle brightness with two-photon excitation than commercial Nile Red-loaded FluoSpheres of 39 nm hydrodynamic diameter. The obtained ultrabright pluronic-coated NPs enabled direct single-particle tracking in vessels of mice brains by two-photon intravital microscopy for at least 1 h, whereas noncoated NPs were rapidly eliminated from the circulation. Following brain injury or neuroinflammation, which can open the blood-brain barrier, extravasation of NPs was successfully monitored. Moreover, we demonstrated tracking of individual NPs from meningeal vessels until their uptake by meningeal macrophages. Thus, single NPs can be tracked in animals in real time in vivo in different brain compartments and their dynamics visualized with subcellular resolution.

Progressive Histopathological Damage Occurring Up to One Year after Experimental Traumatic Brain Injury Is Associated with Cognitive Decline and Depression-Like Behavior.

Increasing clinical and experimental evidence suggests that traumatic brain injury (TBI) is associated with progressive histopathological damage. The aim of the current study was to characterize the time course of motor function, memory performance, and depression-like behavior up to 1 year after experimental TBI, and to correlate these changes to histopathological outcome. Male C57BL/6N mice underwent controlled cortical impact (CCI) or sham operation, and histopathological outcome was evaluated 15 min, 24 h, 1 week, or 1, 3, 6, or 12 months thereafter (n = 12 animals per time point). Motor function, depression-like behavior, and memory function were evaluated concomitantly, and magnetic resonance imaging (MRI) was repeatedly performed. Naïve mice (n = 12) served as an unhandled control group. Injury volume almost doubled within 1 year after CCI (p = 0.008) and the ipsilateral hemisphere became increasingly atrophic (p < 0.0001). Progressive tissue loss was observed in the corpus callosum (p = 0.007) and the hippocampus (p = 0.004) together with hydrocephalus formation (p < 0.0001). Motor function recovered partially after TBI, but 6 months after injury progressive depression-like behavior (p < 0.0001) and loss of memory function (p < 0.0001) were observed. The present study demonstrates that delayed histopathological damage that occurs over months after brain injury is followed by progressive depression and memory loss, changes also observed after TBI in humans. Hence, experimental TBI models in mice replicate long-term sequelae of brain injury such as post-traumatic dementia and depression.

Inadequate food and water intake determine mortality following stroke in mice.

Experimental stroke models producing clinically relevant functional deficits are often associated with high mortality. Because the mechanisms that underlie post-stroke mortality are largely unknown, results obtained using these models are often difficult to interpret, thereby limiting their translational potential. Given that specific forms of post-stroke care reduce mortality in patients, we hypothesized that inadequate food and water intake may underlie mortality following experimental stroke. C57BL/6 mice were subjected to 1 h of intraluminal filament middle cerebral artery occlusion. Nutritional support beginning on the second day after filament middle cerebral artery occlusion reduced the 14-day mortality rate from 59% to 15%. The surviving mice in the post-stroke support group had the same infarct size as non-surviving control mice, suggesting that post-stroke care was not neuroprotective and that inadequate food and/or water intake are the main reasons for filament middle cerebral artery occlusion-induced mortality. This notion was supported by the presence of significant hypoglycemia, ketonemia, and dehydration in control mice. Taken together, these data suggest that post-filament middle cerebral artery occlusion mortality in mice is not primarily caused by ischemic brain damage, but secondarily by inadequate food and/or water intake. Thus, providing nutritional support following filament middle cerebral artery occlusion greatly minimizes mortality bias and allows the study of long-term morphological and functional sequelae of stroke in mice.

Shrinkage-mediated imaging of entire organs and organisms using uDISCO.

Recent tissue-clearing approaches have become important alternatives to standard histology approaches. However, light scattering in thick tissues and the size restrictions on samples that can be imaged with standard light-sheet microscopy pose limitations for analyzing large samples such as an entire rodent body. We developed 'ultimate DISCO' (uDISCO) clearing to overcome these limitations in volumetric imaging. uDISCO preserves fluorescent proteins over months and renders intact organs and rodent bodies transparent while reducing their size up to 65%. We used uDISCO to image neuronal connections and vasculature from head to toe over 7 cm and to perform unbiased screening of transplanted stem cells within the entire body of adult mice. uDISCO is compatible with diverse labeling methods and archival human tissue, and it can readily be used in various biomedical applications to study organization of large organ systems throughout entire organisms.

Pericytes are involved in the pathogenesis of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

OBJECTIVE: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common inherited small-vessel disease, is associated with vascular aggregation of mutant Notch3 protein, dysfunction of cerebral vessels, and dementia. Pericytes, perivascular cells involved in microvascular function, express Notch3. Therefore, we hypothesize that these cells may play a role in the pathogenesis of CADASIL. METHODS: Two-, 7-, and 12-month-old CADASIL mutant mice (TgNotch3(R169C) ) and wild-type controls were examined regarding Notch3 aggregation in pericytes, the coverage of cerebral vessels by pericytes, pericyte numbers, capillary density, blood-brain barrier (BBB) integrity, astrocytic end-feet, and the expression of astrocytic gap junction and endothelial adherens junction protein using immunostaining and Western blot analysis. In addition, we examined cerebrovascular CO2 reactivity using laser Doppler fluxmetry and in vivo microscopy. RESULTS: With increasing age, mutated Notch3 aggregated around pericytes and smooth muscle cells. Notch3 aggregation caused significant reduction of pericyte number and coverage of capillaries by pericyte processes (p < 0.01). These changes were associated with detachment of astrocytic end-feet from cerebral microvessels, leakage of plasma proteins, reduction in expression of endothelial adherens junction protein, and reduced microvascular reactivity to CO2 . Smooth muscle cells were not affected by Notch3 accumulation. INTERPRETATION: Our results show that pericytes are the first cells affected by Notch3 aggregation in CADASIL mice. Pericyte pathology causes opening of the BBB and microvascular dysfunction. Therefore, protecting pericytes may represent a novel therapeutic strategy for vascular dementia.

Dysfunction of mouse cerebral arteries during early aging.

Aging leads to a gradual decline in the fidelity of cerebral blood flow (CBF) responses to neuronal activation, resulting in an increased risk for stroke and dementia. However, it is currently unknown when age-related cerebrovascular dysfunction starts or which vascular components and functions are first affected. The aim of this study was to examine the function of microcirculation throughout aging in mice. Microcirculation was challenged by inhalation of 5% and 10% CO2 or by forepaw stimulation in 6-week, 8-month, and 12-month-old FVB/N mice. The resulting dilation of pial vessels and increase in CBF was measured by intravital fluorescence microscopy and laser Doppler fluxmetry, respectively. Neurovascular coupling and astrocytic endfoot Ca(2+) were measured in acute brain slices from 18-month-old mice. We did not reveal any changes in CBF after CO2 reactivity up to an age of 12 months. However, direct visualization of pial vessels by in vivo microscopy showed a significant, age-dependent loss of CO2 reactivity starting at 8 months of age. At the same age neurovascular coupling was also significantly affected. These results suggest that aging does not affect cerebral vessel function simultaneously, but starts in pial microvessels months before global changes in CBF are detectable.

Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury.

After central nervous system (CNS) injury, inhibitory factors in the lesion scar and poor axon growth potential prevent axon regeneration. Microtubule stabilization reduces scarring and promotes axon growth. However, the cellular mechanisms of this dual effect remain unclear. Here, delayed systemic administration of a blood-brain barrier-permeable microtubule-stabilizing drug, epothilone B (epoB), decreased scarring after rodent spinal cord injury (SCI) by abrogating polarization and directed migration of scar-forming fibroblasts. Conversely, epothilone B reactivated neuronal polarization by inducing concerted microtubule polymerization into the axon tip, which propelled axon growth through an inhibitory environment. Together, these drug-elicited effects promoted axon regeneration and improved motor function after SCI. With recent clinical approval, epothilones hold promise for clinical use after CNS injury.

Microglia in action: how aging and injury can change the brain's guardians.

Neuroinflammation, the inflammatory response in the central nervous system (CNS), is a major determinant of neuronal function and survival during aging and disease progression. Microglia, as the resident tissue-macrophages of the brain, provide constant support to surrounding neurons in healthy brain. Upon any stress signal (such as trauma, ischemia, inflammation) they are one of the first cells to react. Local and/or peripheral signals determine microglia stress response, which can vary within a continuum of states from beneficial to detrimental for neuronal survival, and can be shaped by aging and previous insults. In this review, we discuss the roles of microglia upon an ischemic or traumatic injury, and give our perspective how aging may contribute to microglia behavior in the injured brain. We speculate that a deeper understanding of specific microglia identities will pave the way to develop more potent therapeutics to treat the diseases of aging brain.

A simple method for 3D analysis of immunolabeled axonal tracts in a transparent nervous system.

Clearing techniques have been developed to transparentize mouse brains, thereby preserving 3D structure, but their complexity has limited their use. Here, we show that immunolabeling of axonal tracts followed by optical clearing with solvents (3DISCO) and light-sheet microscopy reveals brain connectivity in mouse embryos and postnatal brains. We show that the Robo3 receptor is selectively expressed by medial habenula axons forming the fasciculus retroflexus (FR) and analyzed the development of this commissural tract in mutants of the Slit/Robo and DCC/Netrin pathways. Netrin-1 and DCC are required to attract FR axons to the midline, but the two mutants exhibit specific and heterogeneous axon guidance defects. Moreover, floor-plate-specific deletion of Slit ligands with a conditional Slit2 allele perturbs not only midline crossing by FR axons but also their anteroposterior distribution. In conclusion, this method represents a unique and powerful imaging tool to study axonal connectivity in mutant mice.

ADF/cofilin-mediated actin retrograde flow directs neurite formation in the developing brain.

Neurites are the characteristic structural element of neurons that will initiate brain connectivity and elaborate information. Early in development, neurons are spherical cells but this symmetry is broken through the initial formation of neurites. This fundamental step is thought to rely on actin and microtubule dynamics. However, it is unclear which aspects of the complex actin behavior control neuritogenesis and which molecular mechanisms are involved. Here, we demonstrate that augmented actin retrograde flow and protrusion dynamics facilitate neurite formation. Our data indicate that a single family of actin regulatory proteins, ADF/Cofilin, provides the required control of actin retrograde flow and dynamics to form neurites. In particular, the F-actin severing activity of ADF/Cofilin organizes space for the protrusion and bundling of microtubules, the backbone of neurites. Our data reveal how ADF/Cofilin organizes the cytoskeleton to drive actin retrograde flow and thus break the spherical shape of neurons.

Three-dimensional imaging of solvent-cleared organs using 3DISCO.

The examination of tissue histology by light microscopy is a fundamental tool for investigating the structure and function of organs under normal and disease states. Many current techniques for tissue sectioning, imaging and analysis are time-consuming, and they present major limitations for 3D tissue reconstruction. The introduction of methods to achieve the optical clearing and subsequent light-sheet laser scanning of entire transparent organs without sectioning represents a major advance in the field. We recently developed a highly reproducible and versatile clearing procedure called 3D imaging of solvent-cleared organs, or 3DISCO, which is applicable to diverse tissues including brain, spinal cord, immune organs and tumors. Here we describe a detailed protocol for performing 3DISCO and present its application to various microscopy techniques, including example results from various mouse tissues. The tissue clearing takes as little as 3 h, and imaging can be completed in ∼45 min. 3DISCO is a powerful technique that offers 3D histological views of tissues in a fraction of the time and labor required to complete standard histology studies.

Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury.

Studying regeneration in the central nervous system (CNS) is hampered by current histological and imaging techniques because they provide only partial information about axonal and glial reactions. Here we developed a tetrahydrofuran-based clearing procedure that renders fixed and unsectioned adult CNS tissue transparent and fully penetrable for optical imaging. In large spinal cord segments, we imaged fluorescently labeled cells by 'ultramicroscopy' and two-photon microscopy without the need for histological sectioning. We found that more than a year after injury growth-competent axons regenerated abundantly through the injury site. A few growth-incompetent axons could also regenerate when they bypassed the lesion. Moreover, we accurately determined quantitative changes of glial cells after spinal cord injury. Thus, clearing CNS tissue enables an unambiguous evaluation of axon regeneration and glial reactions. Our clearing procedure also renders other organs transparent, which makes this approach useful for a large number of preclinical paradigms.