Ischemic stroke is one of the leading causes of long-term disability and death in developed and developing countries. As emerging disease, stroke related mortality and morbidity is going to step up in the next decades. This is both due to the poor identification of risk factors and persistence of unhealthy habits, as well as to the aging of the population. To counteract the estimated increase in stroke incidence, it is of primary importance to identify risk factors, study their effects, to promote primary and secondary prevention, and to extend the therapeutic repertoire that is currently limited to the very first hours after stroke. While epidemiologic studies in the human population are essential to identify emerging risk factors, adequate animal models represent a fundamental tool to dissect stroke risk factors to their molecular mechanism and to find efficacious therapeutic strategies for this complex multi- factorial disorder. The present review is organized into two parts: the first part deals with the animal models that have been developed to study stroke and its related risk factors and the second part analyzes the specific stroke models. These models represent an indispensable tool to investigate the mechanisms of cerebral injury and to develop novel therapies.

Cerebral ischemia stimulates endogenous neurogenesis. However, the functional relevance of this phenomenon remains unclear because of poor survival and low neuronal differentiation rates of newborn cells. Therefore, further studies on mechanisms regulating neurogenesis under ischemic conditions are required, among which ephrin-ligands and ephrin-receptors (Eph) are an interesting target. Although Eph/ephrin proteins like ephrin-B3 are known to negatively regulate neurogenesis under physiological conditions, their role in cerebral ischemia is largely unknown. We therefore studied neurogenesis, brain injury and functional outcome in ephrin-B3(-/-) (knockout) and ephrin-B3(+/+) (wild-type) mice submitted to cerebral ischemia. Induction of stroke resulted in enhanced cell proliferation and neuronal differentiation around the lesion site of ephrin-B3(-/-) compared to ephrin-B3(+/+) mice. However, prominent post-ischemic neurogenesis in ephrin-B3(-/-) mice was accompanied by significantly increased ischemic injury and motor coordination deficits that persisted up to 4 weeks. Ischemic injury in ephrin-B3(-/-) mice was associated with a caspase-3-dependent activation of the signal transducer and activator of transcription 1 (STAT1). Whereas inhibition of caspase-3 had no effect on brain injury in ephrin-B3(+/+) animals, infarct size in ephrin-B3(-/-) mice was strongly reduced, suggesting that aggravated brain injury in these animals might involve a caspase-3-dependent activation of STAT1. In conclusion, post-ischemic neurogenesis in ephrin-B3(-/-) mice is strongly enhanced, but fails to contribute to functional recovery because of caspase-3-mediated aggravation of ischemic injury in these animals. Our results suggest that ephrin-B3 might be an interesting target for overcoming some of the limitations of further cell-based therapies in stroke.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors are widely used for secondary stroke prevention. Besides their lipid-lowering activity, pleiotropic effects on neuronal survival, angiogenesis, and neurogenesis have been described. In view of these observations, we were interested whether HMG-CoA reductase inhibition in the post-acute stroke phase promotes neurological recovery, peri-lesional, and contralesional neuronal plasticity. We examined effects of the HMG-CoA reductase inhibitor rosuvastatin (0.2 or 2.0 mg/kg/day i.c.v.), administered starting 3 days after 30 min of middle cerebral artery occlusion for 30 days. Here, we show that rosuvastatin treatment significantly increased the grip strength and motor coordination of animals, promoted exploration behavior, and reduced anxiety. It was associated with structural remodeling of peri-lesional brain tissue, reflected by increased neuronal survival, enhanced capillary density, and reduced striatal and corpus callosum atrophy. Increased sprouting of contralesional pyramidal tract fibers crossing the midline in order to innervate the ipsilesional red nucleus was noticed in rosuvastatin compared with vehicle-treated mice, as shown by anterograde tract tracing experiments. Western blot analysis revealed that the abundance of HMG-CoA reductase was increased in the contralesional hemisphere at 14 and 28 days post-ischemia. Our data support the idea that HMG-CoA reductase inhibition promotes brain remodeling and plasticity far beyond the acute stroke phase, resulting in neurological recovery.

In the last decade a vast number of animal studies have produced overwhelming evidence that exercise not only compensates for memory loss by increasing brain plasticity and cognitive reserve but also directly counteracts Alzheimer-like pathology when provided before disease onset or in early disease stages. But so far, there is little knowledge about therapeutic effects of training when started in advanced disease stages. In the present study we show that following seven months of sedentary life style five months of wheel running, started four months after disease onset was still able to mitigate at least some aspects of the full-blown Alzheimer's pathology in TgCRND8 mice. Late running had mild but significant effects on structural plasticity by increasing the dendritic complexity. It further reduced beta-amyloid (Aβ) plaque burden and enhanced Aβ clearance across the blood-brain barrier, along with attenuating microgliosis, inflammation, oxidative stress, and autophagy deficits, resulting in better memory performance and less agitation. However, unlike early exercise, late running did not affect abnormal amyloid precursor protein metabolism, tau pathology, or angiogenesis. These results allow concluding that it is never too late to counteract Alzheimer's disease with physical training but the earlier the intervention starts, the more pronounced is the therapeutic potential.

Background and Purpose- Poststroke, neuronal excitability is tonically reduced in peri-infarct tissue via inhibitory influences of extrasynaptic GABAA receptors. We hypothesized that GABAA α5 blockade by the competitive antagonist S44819 enhances postischemic neurological recovery, brain remodeling, and neuroplasticity. Methods- In an explorative study followed by a confirmation study, male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. Starting 72 hours poststroke, vehicle or S44819 (3 or 10 mg/kg, BID) was delivered orally for 28 days. Neurological recovery, perilesional tissue remodeling, and contralesional pyramidal tract plasticity were evaluated for 42 days, that is, 14 days after completion of S44819 delivery. Results- S44819, delivered at 10 but not 3 mg/kg, persistently improved motor coordination and spatial memory in both studies. Striatal atrophy was reduced by 10 mg/kg S44819 at 42 days post-treatment onset, and neuronal long-term survival in the peri-infarct striatum was increased. Delayed neuroprotection was associated with reduced peri-infarct astrogliosis, increased peri-infarct brain capillary density, and increased neural precursor cell proliferation and differentiation in proximity to the ipsilesional subventricular zone. Contralesional pyramidal tract plasticity, evaluated by anterograde tract tracing at the level of the red nucleus, was not influenced by S44819. Concentrations of neurotrophic (brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor) and angiogenic (vascular endothelial growth factor and basic fibroblast growth factor) growth factors were elevated by 10 mg/kg S44819 in peri-infarct but not contralesional brain tissue. Conclusions- Our data demonstrate that S44819 enhances neurological recovery and peri-infarct brain remodeling in the postacute stroke phase.

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

Hypoxia inducible factors (HIFs) mediate angiogenesis via up-regulation of various pro-angiogenic factors (particularly VEGF) in response to hypoxia. Here, we report that hypoxia unexpectedly induced robust production of the pro-inflammatory factor TNFα by endothelial cells (ECs), suggesting an autocrine loop that in turn activated HIFs via an NF-κB-dependent process, resulting in production of VEGF and thereby promotion of angiogenesis. In contrast, low-density lipoprotein (LDL) prevented expression of HIFs in ECs exposed to either hypoxia or TNFα, while knockdown of either HIF-1α or HIF-2α strikingly attenuated hypoxia-induced production of VEGF by ECs as well as EC colony formation and tube formation. Significantly, LDL attenuated hypoxia-induced angiogenesis by disrupting the TNFα/NF-κB/HIF/VEGF signaling cascade via down-regulation of the TNF receptor TNF-R1, rather than TNFα itself, and multiple key components of both canonical and non-canonical NF-κB pathways. By doing so, LDL was able to either inhibit or down-regulate a wide spectrum of HIF-dependent pro-angiogenic downstream targets and signals. Together, these findings argue existence of a self-regulatory TNFα/NF-κB/HIF/VEGF signaling network in ECs, which mediates and fine-tones angiogenesis, at least in response to hypoxia. They also suggest that LDL impairs angiogenesis by disrupting this network, which might represent a novel mechanism underlying anti-angiogenic property of LDL.

Malnutrition predisposes to poor stroke outcome. In animal models, undernutrition protected against ischemic injury in some, but not in other studies. In view of diverse stroke models and food restriction paradigms, the consequences of undernutrition are poorly understood. Herein, we exposed mice to energy-reduced and protein-energy-reduced diets for 7-30 days and subsequently induced intraluminal middle cerebral artery occlusion. Undernutrition phase dependently influenced ischemic injury. Short-lasting 7 days of protein-energy undernutrition, but not energy undernutrition, decreased post-ischemic brain leukocyte infiltration and microglial activation and reduced brain Il-1β mRNA, but did not protect against ischemic injury. Fourteen days of energy and protein-energy undernutrition, on the other hand, reduced ischemic injury despite absence of anti-inflammatory effects. Anti-oxidant genes (Sod-1, Sod-2, and Cat mRNAs) were regulated in the liver and, to a lesser extent, the ischemic brain, indicating an adapted, compensated stage. Conversely, 30 days of energy and protein-energy undernutrition caused progressive animal exhaustion associated with post-ischemic hypoperfusion, rise of metabolic markers (Sirt-1 and Glut-1 mRNAs, Sirt-1 protein) in the ischemic brain, and reregulation of pro- and anti-oxidant markers (now also Nox-4 and Gpx-3 mRNAs) in the liver. In the latter condition, no neuroprotection was noted. Our study suggests an adaptation of metabolic systems that provides neuroprotection in a circumscribed time window.

Cardioprotection by salvage of the infarct-affected myocardium is an unmet yet highly desired therapeutic goal. To develop new dedicated therapies, experimental myocardial ischemia/reperfusion (I/R) injury would require methods to simultaneously characterize extent and localization of the damage and the ensuing inflammatory responses in whole hearts over time. Here we present a three-dimensional (3D), simultaneous quantitative investigation of key I/R injury-components by combining bleaching-augmented solvent-based non-toxic clearing (BALANCE) using ethyl cinnamate (ECi) with light sheet fluorescence microscopy. This allows structural analyses of fluorescence-labeled I/R hearts with exceptional detail. We discover and 3D-quantify distinguishable acute and late vascular I/R damage zones. These contain highly localized and spatially structured neutrophil infiltrates that are modulated upon cardiac healing. Our model demonstrates that these characteristic I/R injury patterns can detect the extent of damage even days after the ischemic index event hence allowing the investigation of long-term recovery and remodeling processes.

Adhesion molecules are key elements in stroke-induced brain injury by regulating the migration of effector immune cells from the circulation to the lesion site. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an adhesion molecule highly expressed on endothelial cells and leukocytes, which controls the final steps of trans-endothelial migration. A functional role for PECAM-1 in post-ischemic brain injury has not yet been demonstrated.

Extracellular vesicles (EVs) derived from neural progenitor cells enhance poststroke neurological recovery, albeit the underlying mechanisms remain elusive. Since previous research described an enhanced poststroke integrity of the blood-brain barrier (BBB) upon systemic transplantation of neural progenitor cells, we examined if neural progenitor cell-derived EVs affect BBB integrity and which cellular mechanisms are involved in the process. Approach and Results: Using in vitro models of primary brain endothelial cell (EC) cultures as well as co-cultures of brain ECs (ECs) and astrocytes exposed to oxygen glucose deprivation, we examined the effects of EVs or vehicle on microvascular integrity. In vitro data were confirmed using a mouse transient middle cerebral artery occlusion model. Cultured ECs displayed increased ABCB1 (ATP-binding cassette transporter B1) levels when exposed to oxygen glucose deprivation, which was reversed by treatment with EVs. The latter was due to an EV-induced inhibition of the NF-κB (nuclear factor-κB) pathway. Using a BBB co-culture model of ECs and astrocytes exposed to oxygen glucose deprivation, EVs stabilized the BBB and ABCB1 levels without affecting the transcellular electrical resistance of ECs. Likewise, EVs yielded reduced Evans blue extravasation, decreased ABCB1 expression as well as an inhibition of the NF-κB pathway, and downstream matrix metalloproteinase 9 (MMP-9) activity in stroke mice. The EV-induced inhibition of the NF-κB pathway resulted in a poststroke modulation of immune responses.

Until recently, the visualization of cerebral microvessels was hampered by the fact that only short segments of vessels could be evaluated in brain sections by histochemistry. These limitations have been overcome by light sheet microscopy, which allows the 3D analysis of microvasculature in cleared brains. A major limitation of light sheet microscopy is that antibodies do not sufficiently penetrate cleared brains. We herein describe a technique of reverse clearing and rehydration, which after microvascular network analysis allows brain sectioning and immunohistochemistry employing a broad set of antibodies. Performing light sheet microscopy on brains of mice exposed to intraluminal middle cerebral artery occlusion (MCAO), we show that in the early phase of microvascular remodeling branching point density was markedly reduced, more strongly than microvascular length. Brain infarcts in light sheet microscopy were sharply demarcated by their autofluorescence signal, closely corresponding to brain infarcts revealed by Nissl staining. Neuronal survival, leukocyte infiltration, and astrocytic reactivity could be evaluated by immunohistochemistry in rehydrated brains, as shown in direct comparisons with non-cleared brains. Immunohistochemistry revealed microthrombi in ischemic microvessels that were likely responsible for the marked branching point loss. The balance between microvascular thrombosis and remodeling warrants further studies at later time-points after stroke.

Grafted mesenchymal stem cells (MSCs) yield neuroprotection in preclinical stroke models by secreting extracellular vesicles (EVs). The neuroprotective cargo of EVs, however, has not yet been identified. To investigate such cargo and its underlying mechanism, primary neurons were exposed to oxygen-glucose-deprivation (OGD) and cocultured with adipose-derived MSCs (ADMSCs) or ADMSC-secreted EVs. Under such conditions, both ADMSCs and ADMSC-secreted EVs significantly reduced neuronal death. Screening for signalling cascades being involved in the interaction between ADMSCs and neurons revealed a decreased autophagic flux as well as a declined p53-BNIP3 activity in neurons receiving either treatment paradigm. However, the aforementioned effects were reversed when ADMSCs were pretreated with the inhibitor of exosomal secretion GW4869 or when Hrs was knocked down. In light of miR-25-3p being the most highly expressed miRNA in ADMSC-EVs interacting with the p53 pathway, further in vitro work focused on this pathway. Indeed, a miR-25-3p oligonucleotide mimic reduced cell death, whereas the anti-oligonucleotide increased autophagic flux and cell death by modulating p53-BNIP3 signalling in primary neurons exposed to OGD. Likewise, native ADMSC-EVs but not EVs obtained from ADMSCs pretreated with the anti-miR-25-3p oligonucleotide (ADMSC-EVsanti-miR-25-3p) confirmed the aforementioned in vitro observations in C57BL/6 mice exposed to cerebral ischemia. The infarct size was reduced, and neurological recovery was increased in mice treated with native ADMSC-EVs when compared to ADMSC-EVsanti-miR-25-3p. ADMSCs induce neuroprotection by improved autophagic flux through secreted EVs containing miR-25-3p. Hence, our work uncovers a novel key factor in naturally secreted ADMSC-EVs for the regulation of autophagy and induction of neuroprotection in a preclinical stroke model.

Calorie restriction confers post-ischemic neuroprotection, when administered in a defined time window before ischemic stroke. How a hypocaloric diet influences stroke recovery when initiated after stroke has not been investigated. Male C57BL6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. Immediately post-ischemia, mice were randomized to two groups receiving moderately hypocaloric (2286 kcal/kg food) or normocaloric (3518 kcal/kg) diets ad libitum. Animals were sacrificed at 3 or 56 days post-ischemia (dpi). Besides increased low density lipoprotein at 3 days and reduced alanine aminotransferase and increased urea at 56 days, no alterations of plasma markers were found in ischemic mice on hypocaloric diet. Body weight mildly decreased over 56 dpi by 7.4%. Hypocaloric diet reduced infarct volume in the acute stroke phase at 3 dpi and decreased brain atrophy, increased neuronal survival and brain capillary density in peri-infarct striatum and reduced motor coordination impairment in tight rope tests in the post-acute stroke phase over up to 56 dpi. The abundance of brain-derived neurotrophic factor, the NAD-dependent deacetylase and longevity protein sirtuin-1, the anti-oxidant glutathione peroxidase-3, and the ammonium detoxifier glutamine synthetase in the peri-infarct brain tissue was increased by hypocaloric diet. This study shows that a moderately hypocaloric diet that is initiated after stroke confers long-term neuroprotection and promotes peri-infarct brain remodeling.

As an endogenous activator of toll-like receptor-4 (Tlr4), the extracellular matrix glycoprotein tenascin-C (TnC) regulates chemotaxis, phagocytosis and proinflammatory cytokine production in microglia. The role of TnC for ischemic brain injury, post-ischemic immune responses and stroke recovery has still not been evaluated. By comparing wild type and TnC-/- mice exposed to transient intraluminal middle cerebral artery occlusion (MCAO), we examined the effects of TnC deficiency for ischemic injury, neurological deficits, microglia/macrophage activation and brain leukocyte infiltration using behavioural tests, histochemical studies, Western blot, polymerase chain reaction and flow cytometry. Histochemical studies revealed that TnC was de novo expressed in the ischemic striatum, which contained the infarct core, and overlapped with the area of strongest accumulation of Iba1 + microglia/macrophages. TnC deficiency increased overall Iba1 immunoreactivity in the perilesional cortex, suggesting that TnC might restrict the distribution of microglial cells to the infarct core. By analysing microglial morphology in 3D we found that the post-ischemic loss of microglial cell territory, branching and volume at 3 and 7 days post-ischemia was amplified in the brains of TnC deficient compared with wild type mice. Microglial cell number was not different between genotypes. Hence, TnC deficiency reduced tissue surveillance by microglial cells. Concomitantly, the number of infiltrating leukocytes and, more specifically, T cells was increased in the ischemic brain parenchyma of TnC deficient compared with wild type mice. Ischemic injury and neurological deficits were not affected by TnC deficiency. We propose that the reduced microglia surveillance in TnC deficient mice might favour leukocyte accumulation in the ischemic brain.

Adipose-derived mesenchymal stem cells markedly attenuated brain infarct size and improved neurological function in rats. The mechanisms for neuronal cell death have previously been defined in stress states to suggest that an influx of calcium ions into the neurons activates calpain cleavage of p35 into p25 forming a hyperactive complex that induces cell death. Now we report that p5, a 24-residue peptide derived from p35, offers protection to neurons and endothelial cells in vitro. In vivo administration of human adipose-derived mesenchymal stem cells (hADMSCs) loaded with this therapeutic peptide to post-stroke rats had no effect on the infarct volume. Nevertheless, the treatment led to improvement in functional recovery in spatial learning and memory (water maze), bilateral coordination and sensorimotor function (rotating pole), and asymmetry of forelimb usage (cylinder test). However, the treatment may not impact on cutaneous sensitivity (adhesive tape removal test). In addition, the double immunofluorescence with human cell-specific antibodies revealed that the number of surviving transplanted cells was higher in the peri-infarcted area of animals treated with hADMSCs + P5 than that in hADMSC-treated or control animals, concomitant with reduced number of phagocytic, annexin3-positive cells in the peri-infarcted region. However, the combination therapy did not increase the vascular density in the peri-infarcted area after stroke. In conclusion, administration of hADMSC-loaded p5 peptide to post-stroke rats created conditions that supported survival of drug-loaded hADMSCs after cerebral ischemia, suggesting its therapeutic potential in patients with stroke.

Inhibitory control is essential for the regulation of neuronal network activity, where excitatory and inhibitory synapses can act synergistically, reciprocally, and antagonistically. Sustained excitation-inhibition (E-I) balance, therefore, relies on the orchestrated adjustment of excitatory and inhibitory synaptic strength. While growing evidence indicates that the brain's extracellular matrix (ECM) is a crucial regulator of excitatory synapse plasticity, it remains unclear whether and how the ECM contributes to inhibitory control in neuronal networks. Here we studied the simultaneous changes in excitatory and inhibitory connectivity after ECM depletion. We demonstrate that the ECM supports the maintenance of E-I balance by retaining inhibitory connectivity. Quantification of synapses and super-resolution microscopy showed that depletion of the ECM in mature neuronal networks preferentially decreases the density of inhibitory synapses and the size of individual inhibitory postsynaptic scaffolds. The reduction of inhibitory synapse density is partially compensated by the homeostatically increasing synaptic strength via the reduction of presynaptic GABAB receptors, as indicated by patch-clamp measurements and GABAB receptor expression quantifications. However, both spiking and bursting activity in neuronal networks is increased after ECM depletion, as indicated by multi-electrode recordings. With computational modelling, we determined that ECM depletion reduces the inhibitory connectivity to an extent that the inhibitory synapse scaling does not fully compensate for the reduced inhibitory synapse density. Our results indicate that the brain's ECM preserves the balanced state of neuronal networks by supporting inhibitory control via inhibitory synapse stabilization, which expands the current understanding of brain activity regulation.

Infection is a major reason for poor stroke outcomes, and sepsis is a major cause of stroke-elated deaths. We herein examined whether NMDA receptor blockade, which was reported to exert anti-inflammatory actions, protects against the deleterious consequences of lipopolysaccharide (LPS)-induced sepsis-like state in adult male NMRI mice exposed to transient intraluminal middle cerebral artery occlusion (MCAO). At 24 h post-ischemia, vehicle or Escherichia coli LPS (2 or 4 mg/kg) was intraperitoneally administered, whereas 30 min later vehicle or ketamine (10 mg/kg), which is a non-competitive NMDA receptor antagonist, was intraperitoneally applied. Delivery of LPS at a dosage of 4 mg/kg induced a sepsis-like state characterized by a rectal temperature reduction by ∼4.0°C, increased neurological deficits in Clark score, cylinder and open-field tests, increased brain infarct volume and reduced neuronal survival in the previously ischemic tissue. Notably, additional treatment with ketamine (10 mg/kg) significantly attenuated the sepsis-associated rectal temperature reduction by ∼1.5°C, reduced neurological deficits, reduced infarct volume, and promoted neuronal survival. Ketamine alone did not influence infarct volume or neurological deficits. Real-time PCR data analysis showed that GFAP, CD86, CD206, IL-1β, and IL-10 mRNA levels were significantly increased in ischemic brains of LPS-treated compared with vehicle-treated mice. Additional treatment with ketamine significantly decreased IL-1β and IL-10, but not GFAP, CD86, and CD206 mRNA levels. Our data show that ketamine at a dose that on its own does not confer neuroprotection reverses the adverse effects of LPS-induced sepsis-like state post-ischemia, presumably via immunomodulatory actions.

Light-sheet fluorescence microscopy (LSFM) can produce high-resolution tomograms of tissue vasculature with high accuracy. However, data processing and analysis is laborious due to the size of the datasets. Here, we introduce VesselExpress, an automated software that reliably analyzes six characteristic vascular network parameters including vessel diameter in LSFM data on average computing hardware. VesselExpress is ∼100 times faster than other existing vessel analysis tools, requires no user interaction, and integrates batch processing and parallelization. Employing an innovative dual Frangi filter approach, we show that obesity induces a large-scale modulation of brain vasculature in mice and that seven other major organs differ strongly in their 3D vascular makeup. Hence, VesselExpress transforms LSFM from an observational to an analytical working tool.

Repetitive transcranial magnetic stimulation (rTMS) has gained interest as a non-invasive treatment for stroke based on the data promoting its effects on functional recovery. However, the exact action mechanisms by which the rTMS exert beneficial effects in cellular and molecular aspect are largely unknown. To elucidate the effects of high- and low-frequency rTMS in the acute-ischemic brain, we examined how rTMS influences injury development, cerebral blood flow (CBF), DNA fragmentation, neuronal survival, pro- and anti-apoptotic protein activations after 30 and 90 min of focal cerebral ischemia. In addition, inflammation, angiogenesis, growth factors and axonal outgrowth related gene expressions, were analyzed. Furthermore, we have investigated the effects of rTMS on post-acute ischemic brain, particularly on spontaneous locomotor activity, perilesional tissue remodeling, axonal sprouting of corticobulbar tracts, glial scar formation and cell proliferation, in which rTMS was applied starting 3 days after the stroke onset for 28 days. In the high-frequency rTMS received animals reduced DNA fragmentation, infarct volume and improved CBF were observed, which were associated with increased Bcl-xL activity and reduced Bax, caspase-1, and caspase-3 activations. Moreover, increased angiogenesis, growth factors; and reduced inflammation and axonal sprouting related gene expressions were observed. These results correlated with reduced microglial activation, neuronal degeneration, glial scar formation and improved functional recovery, tissue remodeling, contralesional pyramidal tract plasticity and neurogenesis in the subacute rTMS treated animals. Overall, we propose that high-frequency rTMS in stroke patients can be used to promote functional recovery by inducing the endogenous repair and recovery mechanisms of the brain.