Two-photon Laser Scanning Microscopy Research Articles

PRMT7 can prevent neurovascular uncoupling, blood-brain barrier permeability, and mitochondrial dysfunction in repetitive and mild traumatic brain injury

Mild traumatic brain injury (TBI) comprises the largest percentage of TBI-related injuries, with pathophysiological and functional deficits that persist in a subset of TBI patients. In our three-hit paradigm of repetitive and mild traumatic brain injury (rmTBI), we observed neurovascular uncoupling via decreased red blood cell velocity, microvessel diameter, and leukocyte rolling velocity 3 days post-rmTBI via intra-vital two-photon laser scanning microscopy. Furthermore, our data suggest increased blood-brain barrier (BBB) permeability (leakage), with corresponding decrease in junctional protein expression post-rmTBI. Mitochondrial oxygen consumption rates (measured via Seahorse XFe24) were also altered 3 days post-rmTBI, along with disrupted mitochondrial dynamics of fission and fusion. Overall, these pathophysiological findings correlated with decreased protein arginine methyltransferase 7 (PRMT7) protein levels and activity post-rmTBI. Here, we increased PRMT7 levels in vivo to assess the role of the neurovasculature and mitochondria post-rmTBI. In vivo overexpression of PRMT7 using a neuronal specific AAV vector led to restoration of neurovascular coupling, prevented BBB leakage, and promoted mitochondrial respiration, altogether to suggest a protective and functional role of PRMT7 in rmTBI.

Deficits in mitochondrial TCA cycle and OXPHOS precede rod photoreceptor degeneration during chronic HIF activation

BackgroundMajor retinal degenerative diseases, including age-related macular degeneration, diabetic retinopathy and retinal detachment, are associated with a local decrease in oxygen availability causing the formation of hypoxic areas affecting the photoreceptor (PR) cells. Here, we addressed the underlying pathological mechanisms of PR degeneration by focusing on energy metabolism during chronic activation of hypoxia-inducible factors (HIFs) in rod PR.MethodsWe used two-photon laser scanning microscopy (TPLSM) of genetically encoded biosensors delivered by adeno-associated viruses (AAV) to determine lactate and glucose dynamics in PR and inner retinal cells. Retinal layer-specific proteomics, in situ enzymatic assays and immunofluorescence studies were used to analyse mitochondrial metabolism in rod PRs during chronic HIF activation.ResultsPRs exhibited remarkably higher glycolytic flux through the hexokinases than neurons of the inner retina. Chronic HIF activation in rods did not cause overt change in glucose dynamics but an increase in lactate production nonetheless. Furthermore, dysregulation of the oxidative phosphorylation pathway (OXPHOS) and tricarboxylic acid (TCA) cycle in rods with an activated hypoxic response decelerated cellular anabolism causing shortening of rod photoreceptor outer segments (OS) before onset of cell degeneration. Interestingly, rods with deficient OXPHOS but an intact TCA cycle did not exhibit these early signs of anabolic dysregulation and showed a slower course of degeneration.ConclusionTogether, these data indicate an exceeding high glycolytic flux in rods and highlight the importance of mitochondrial metabolism and especially of the TCA cycle for PR survival in conditions of increased HIF activity.

ROS Scavenging Graphene-Based Hydrogel Enhances Type H Vessel Formation and Vascularized Bone Regeneration via ZEB1/Notch1 Mediation.

The regeneration strategy for bone defects is greatly limited by the bone microenvironment, and excessive reactive oxygen species (ROS) seriously hinder the formation of new bone. Reduced graphene oxide (rGO) is expected to meet the requirements because of its ability to scavenge free radicals through electron transfer. Antioxidant hydrogels based on gelatine methacrylate (GM), acrylyl-β-cyclodextrin (Ac-CD), and rGO functionalized with β-cyclodextrin (β-CD) are developed for skull defect regeneration, but the mechanism of how rGO-based hydrogels enhance bone repair remains unclear. In this work, it is confirmed that the GM/Ac-CD/rGO hydrogel has good antioxidant capacity, and promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs). The rGO-based hydrogel affects ZEB1/Notch1 to promote tube formation. Furthermore, two-photon laser scanning microscopy is used to observe the ROS in a skull defect. The rGO-based hydrogel promotes type H vessel formation in a skull defect. In conclusion, the hydrogel neutralizes ROS in the vicinity of a skull defect and stimulates ZEB1/Notch1 to promote the coupling of osteogenesis and angiogenesis, which may be a possible approach for bone regeneration.

Visualization of the pH Response through Autofluorescent Poly(styrene-alt-N-maleimide) Polyelectrolyte Brushes

Polyelectrolyte brushes can undergo reversible conformational transitions in response to changes in environmental pH and ionic strength. Therefore, they offer great potential for the design of “smart” surfaces and surface-based sensing devices. Herein, we report weak acidic polyelectrolyte brushes with pH-dependent autofluorescence based on alternating copolymers of styrene and tailor-made N-maleimides, which exhibit “clusteroluminescence” due to the through-space conjugation of π-chromophoric subfluorophores. Swelling behavior of the polyelectrolyte brushes was evaluated as a function of pH via in-solution atomic force microscopy (AFM) analyses. The correlation between the pH-induced conformational transitions and autofluorescence was confirmed with confocal laser scanning microscopy (CLSM) and two-photon laser scanning microscopy. Poly(styrene-alt-N-maleimide)-based well-defined, stable polyelectrolyte brushes, generating optical signals from conformational changes without conventional fluorophores, may enable the design of sensors and optoelectronic devices.

From static to dynamic: live observation of the support system after ischemic stroke by two photon-excited fluorescence laser-scanning microscopy.

Ischemic stroke is one of the most common causes of mortality and disability worldwide. However, treatment efficacy and the progress of research remain unsatisfactory. As the critical support system and essential components in neurovascular units, glial cells and blood vessels (including the blood-brain barrier) together maintain an optimal microenvironment for neuronal function. They provide nutrients, regulate neuronal excitability, and prevent harmful substances from entering brain tissue. The highly dynamic networks of this support system play an essential role in ischemic stroke through processes including brain homeostasis, supporting neuronal function, and reacting to injuries. However, most studies have focused on postmortem animals, which inevitably lack critical information about the dynamic changes that occur after ischemic stroke. Therefore, a high-precision technique for research in living animals is urgently needed. Two-photon fluorescence laser-scanning microscopy is a powerful imaging technique that can facilitate live imaging at high spatiotemporal resolutions. Two-photon fluorescence laser-scanning microscopy can provide images of the whole-cortex vascular 3D structure, information on multicellular component interactions, and provide images of structure and function in the cranial window. This technique shifts the existing research paradigm from static to dynamic, from flat to stereoscopic, and from single-cell function to multicellular intercommunication, thus providing direct and reliable evidence to identify the pathophysiological mechanisms following ischemic stroke in an intact brain. In this review, we discuss exciting findings from research on the support system after ischemic stroke using two-photon fluorescence laser-scanning microscopy, highlighting the importance of dynamic observations of cellular behavior and interactions in the networks of the brain's support systems. We show the excellent application prospects and advantages of two-photon fluorescence laser-scanning microscopy and predict future research developments and directions in the study of ischemic stroke.

Sex-Specific and Dose-Dependent Effects of Drag-Reducing Polymers on Microcirculation and Tissue Oxygenation in Rats After Traumatic Brain Injury.

Traumatic brain injury (TBI) ultimately leads to a reduction in the cerebral metabolic rate for oxygen due to ischemia. Previously, we showed that 2ppm i.v. of drag-reducing polymers (DRP) improve hemodynamic and oxygen delivery to tissue in a rat model of mild-to-moderate TBI. Here we evaluated sex-specific and dose-dependent effects of DRP on microvascular CBF (mvCBF) and tissue oxygenation in rats after moderate TBI. In vivo two-photon laser scanning microscopy over the rat parietal cortex was used to monitor the effects of DRP on microvascular perfusion, tissue oxygenation, and blood-brain barrier (BBB) permeability. Lateral fluid-percussion TBI (1.5 ATA, 100ms) was induced after baseline imaging and followed by 4h of monitoring. DRP was injected at 1, 2, or 4ppm within 30 min after TBI. Differences between groups were determined using a two-way ANOVA analysis for multiple comparisons and post hoc testing using the Mann-Whitney U test. Moderate TBI progressively decreased mvCBF, leading to tissue hypoxia and BBB degradation in the pericontusion zone (p<0.05). The i.v. injection of DRP increased near-wall flow velocity and flow rate in arterioles, leading to an increase in the number of erythrocytes entering capillaries, enhancing capillary perfusion and tissue oxygenation while protecting BBB in a dose-dependent manner without significant difference between males and females (p<0.01). TBI resulted in an increase in intracranial pressure (20.1±3.2mmHg, p<0.05), microcirculatory redistribution to non-nutritive microvascular shunt flow, and stagnation of capillary flow, all of which were dose-dependently mitigated by DRP. DRP at 4ppm was most effective, with a non-significant trend to better outcomes in female rats.

A novel tool to study bipolar cell responses in glaucoma

AbstractPurpose: Retinal ganglion cell death has been well studied in glaucoma, however how the calcium (Ca2+) dynamics in living inner retinal neurons alter to ocular hypertensive stress is poorly understood. Here we characterized a novel adeno‐associated virus (AAV) mediated Ca2+ sensor to investigate light‐evoked Ca2+ responses in bipolar cells with two‐photon laser scanning microscopy (TPLSM).Methods: To visualize ON bipolar cell specific Ca2+ expression, we bioinformatically designed a serotype 2 AAV vector carrying GCaMP6f, a Ca2+indicator, under human MiniPromoter: Ple265 (PCP2, 986 bp, AAV.Ple265.GCaMP). The AAV.Ple265.GCaMP6 was administered intravitreal to adult mice (2 μl/eye). Two and 4 weeks later (n = 3 mice/group), eyes were collected, and retinal cross sections (16 μm) were prepared for immunohistochemistry using the following primary antibodies: GFP to visualize GCaMP6f, and bipolar cell‐specific markers PCP2 or PKCa. Furthermore, we monitored real time light‐evoked Ca2+ responses in bipolar cell terminals at 4 weeks after the injection and amplitude (ΔF/F0) was calculated.Results: At 2 weeks after the viral injection, GCaMP6f was clearly visualized in bipolar cells, but its expression was limited to the cell body and dendritic trees. Four weeks post‐injection, GCaMP6f expression was abundantly detected in many bipolar cell bodies, dendrites, axons, and axonal terminals. TPLSM allowed visualization of bipolar cell‐specific GCaMP6f expression in axonal terminals in vivo, and light‐evoked sustained ON‐responses were recorded in terminal microdomains (average intensity = 1.25 ΔF/F0).Conclusions: Our study reveals that the AAV.Ple265.GCaMP is a powerful tool to monitor ON bipolar cell specific light‐evoked Ca2+ responses in bipolar cells, and can be potentially used for studies aimed at understanding the response of these neurons to glaucomatous damage.

EXTH-02. ANTI-TUMOR EFFECTS AND IN VIVO DYNAMICS OF EPCAM-DIRECTED CAR T-CELLS FOR BRAIN METASTASES FROM LUNG CANCER

Abstract BACKGROUND Lung cancer patients are at a high risk for brain metastases, and affected patients frequently succumb to their intracranial disease. Chimeric Antigen Receptor (CAR) T-cells emerged as a powerful cell-based immunotherapy for hematological malignancies; however, it remains unclear whether CAR T-cells represent a viable therapeutic avenue for brain metastases. METHODS A fully immunocompetent, orthotopic cerebral metastasis model was established in mice by combining a chronic cranial window with repetitive intracerebral two-photon laser scanning microscopy. This approach enabled the in vivo-characterization of fluorescent CAR T-cells and tumor cells on a single-cell level over weeks. Intraparenchymal injection of EpCAM-expressing Lewis lung carcinoma cells was performed, and EpCAM-directed CAR T-cells (EpCAMCAR T-cells) were injected into the adjacent brain parenchyma after brain tumor formation. RESULTS All mice had visible tumor take with rapidly growing lesions following intracranial tumor cell injection. In mice treated with EpCAMCAR T-cells, we observed substantial CAR T-cell accumulation within the tumor compared to controls treated with undirected T-cells. This was paralleled by lower velocities of EpCAMCAR T-cells, characterizing antitumor cytotoxicity due to ‘immune cell’-‘tumor cell’ contacts. Consequently, treatment with EpCAMCAR T-cells resulted in reduced tumorous growth as determined per in vivo-microscopy (median tumor area on day 10: 1.8 versus 10.8 mm2; p=0.001) and immunohistochemistry of excised brains. However, the number of intratumoral EpCAMCAR T-cells within the tumor markedly decreased during the observation period, pointing towards insufficient persistence. Accordingly, survival was prolonged in mice receiving EpCAMCAR T-cells but long-lasting remission was rare (median survival: 15 versus 13 days; p=0.012). No CNS-specific or systemic toxicities of EpCAMCAR T-cells were encountered. CONCLUSION Our findings indicate that EpCAMCAR T-cells injected into the cerebral parenchyma may safely induce relevant anti-tumor effects in brain metastases from lung cancer. Strategies improving the CAR T-cell persistence within brain metastases are warranted to further boost the therapeutic success.

Cannabidiol Exerts a Neuroprotective and Glia-Balancing Effect in the Subacute Phase of Stroke.

Pharmacological agents limiting secondary tissue loss and improving functional outcomes after stroke are still limited. Cannabidiol (CBD), the major non-psychoactive component of Cannabis sativa, has been proposed as a neuroprotective agent against experimental cerebral ischemia. The effects of CBD mostly relate to the modulation of neuroinflammation, including glial activation. To investigate the effects of CBD on glial cells after focal ischemia in vivo, we performed time-lapse imaging of microglia and astroglial Ca2+ signaling in the somatosensory cortex in the subacute phase of stroke by in vivo two-photon laser-scanning microscopy using transgenic mice with microglial EGFP expression and astrocyte-specific expression of the genetically encoded Ca2+ sensor GCaMP3. CBD (10 mg/kg, intraperitoneally) prevented ischemia-induced neurological impairment, reducing the neurological deficit score from 2.0 ± 1.2 to 0.8 ± 0.8, and protected against neurodegeneration, as shown by the reduction (more than 70%) in Fluoro-Jade C staining (18.8 ± 7.5 to 5.3 ± 0.3). CBD reduced ischemia-induced microglial activation assessed by changes in soma area and total branch length, and exerted a balancing effect on astroglial Ca2+ signals. Our findings indicate that the neuroprotective effects of CBD may occur in the subacute phase of ischemia, and reinforce its strong anti-inflammatory property. Nevertheless, its mechanism of action on glial cells still requires further studies.

OS03.4.A In vivo dynamics and anti-tumor effects of EpCAM-directed CAR T-cells against brain metastases from lung cancer

Abstract Background Lung cancer patients are at a particularly high risk for brain metastases, and a considerable number of affected patients succumb due to their intracranial disease. Chimeric Antigen Receptor (CAR) T-cells emerged as a powerful class of cell-based immunotherapy for hematological malignancies. However, it remains unclear whether CAR T-cells also represent a safe and effective therapeutic option for brain metastases. Material and Methods A fully syngeneic orthotopic cerebral metastasis model in mice was established by combining a chronic cranial window with repetitive intracerebral two-photon laser scanning microscopy. This approach enabled the in vivo characterization of red fluorescent CAR T-cells and tumor cells on a single-cell level over weeks. Intraparenchymal injection of Lewis lung carcinoma cells (expressing the tumor cell-antigen EpCAM) was performed, and CAR T-cells directed against EpCAM (EpCAMCAR T-cells) were injected into the adjacent brain parenchyma after solid brain tumors have formed. Results All mice had visible tumor take four days after the intracranial tumor cell injection, and rapid growth of solitary lesions was seen in the following days. No toxicities mediated by intracranially injected EpCAMCAR T-cells were encountered. In mice treated with EpCAMCAR T-cells, we initially observed substantial CAR T-cell accumulation within the tumor compared to controls (median number: 1700 versus 939 cells/mm2; p = 0.007). This was paralleled by lower velocities of EpCAMCAR T-cells, characterizing T-cell receptor (TCR)-mediated antitumor cytotoxicity due to long-lasting contacts between effector immune cells and tumor cells. Consequently, treatment with EpCAMCAR T-cells resulted in reduced tumorous growth as determined per in vivo microscopy (median tumor area on day 10: 1.8 versus 10.8 mm2; p = 0.001) and immunohistochemistry of excised brains (median tumor volume on day 10: 3.6 versus 33.2 mm3; p = 0.001). However, the number of EpCAM-directed CAR T-cells within the tumor markedly decreased during the observation period, pointing towards insufficient persistence of EpCAM-directed CAR T-cells. In line with these findings, survival was prolonged in mice receiving EpCAMCAR T-cells but long-lasting remission was rare (median survival: 15 versus 13 days; p = 0.012). Conclusion Our findings indicate that CAR T-cells injected into the cerebral parenchyma may induce relevant anti-tumor effects in brain metastases from lung cancer. Due to the translational nature of our study, prospective clinical cohorts appear warranted. Strategies improving the intratumoral persistence of CAR T-cells in brain metastases may be utilized to further boost the success of such promising therapy.

&lt;em&gt;In Vivo&lt;/em&gt; Vascular Permeability Detection in Mouse Submandibular Gland

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

Dysbindin-1A modulation of astrocytic dopamine and basal ganglia dependent behaviors relevant to schizophrenia.

The mechanisms underlying the dichotomic cortical/basal ganglia dopaminergic abnormalities in schizophrenia are unclear. Astrocytes are important non-neuronal modulators of brain circuits, but their role in dopaminergic system remains poorly explored. Microarray analyses, immunohistochemistry, and two-photon laser scanning microscopy revealed that Dys1 hypofunction increases the reactivity of astrocytes, which express only the Dys1A isoform. Notably, behavioral and electrochemical assessments in mice selectively lacking the Dys1A isoform unraveled a more prominent impact of Dys1A in behavioral and dopaminergic/D2 alterations related to basal ganglia, but not cortical functioning. Ex vivo electron microscopy and protein expression analyses indicated that selective Dys1A disruption might alter intracellular trafficking in astrocytes, but not in neurons. In agreement, Dys1A disruption only in astrocytes resulted in decreased motivation and sensorimotor gating deficits, increased astrocytic dopamine D2 receptors and decreased dopaminergic tone within basal ganglia. These processes might have clinical relevance because the caudate, but not the cortex, of patients with schizophrenia shows a reduction of the Dys1A isoform. Therefore, we started to show a hitherto unknown role for the Dys1A isoform in astrocytic-related modulation of basal ganglia behavioral and dopaminergic phenotypes, with relevance to schizophrenia.

Bone Marrow Derived Mesenchymal Stromal Cells Promote Vascularization and Ciliation in Airway Mucosa Tri-Culture Models in Vitro

Patients suffering from irresectable tracheal stenosis often face limited treatment options associated with low quality of life. To date, an optimal tracheal replacement strategy does not exist. A tissue-engineered tracheal substitute promises to overcome limitations such as implant vascularization, functional mucociliary clearance and mechanical stability. In order to advance a tracheal mucosa model recently developed by our group, we examined different supporting cell types in fibrin-based tri-culture with primary human umbilical vein endothelial cells (HUVEC) and primary human respiratory epithelial cells (HRE). Bone marrow-derived mesenchymal stromal cells (BM-MSC), adipose-derived mesenchymal stromal cells (ASC) and human nasal fibroblasts (HNF) were compared regarding their ability to promote mucociliary differentiation and vascularization in vitro. Three-dimensional co-cultures of the supporting cell types with either HRE or HUVEC were used as controls. Mucociliary differentiation and formation of vascular-like structures were analyzed by scanning electron microscopy (SEM), periodic acid Schiff’s reaction (PAS reaction), two-photon laser scanning microscopy (TPLSM) and immunohistochemistry. Cytokine levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-6 (IL6), interleukin-8 (IL8), angiopoietin 1, angiopoietin 2, fibroblast growth factor basic (FGF-b) and placenta growth factor (PIGF) in media supernatant were investigated using LEGENDplex™ bead-based immunoassay. Epithelial morphology of tri-cultures with BM-MSC most closely resembled native respiratory epithelium with respect to ciliation, mucus production as well as expression and localization of epithelial cell markers pan-cytokeratin, claudin-1, α-tubulin and mucin5AC. This was followed by tri-cultures with HNF, while ASC-supported tri-cultures lacked mucociliary differentiation. For all supporting cell types, a reduced ciliation was observed in tri-cultures compared to the corresponding co-cultures. Although formation of vascular-like structures was confirmed in all cultures, vascular networks in BM-MSC-tri-cultures were found to be more branched and extended. Concentrations of pro-angiogenic and inflammatory cytokines, in particular VEGF and angiopoietin 2, revealed to be reduced in tri-cultures compared to co-cultures. With these results, our study provides an important step towards a vascularized and ciliated tissue-engineered tracheal replacement. Additionally, our tri-culture model may in the future contribute to an improved understanding of cell-cell interactions in diseases associated with impaired mucosal function.

A silent two-photon imaging system for studying in vivo auditory neuronal functions

Two-photon laser-scanning microscopy has become an essential tool for imaging neuronal functions in vivo and has been applied to different parts of the neural system, including the auditory system. However, many components of a two-photon microscope, such as galvanometer-based laser scanners, generate mechanical vibrations and thus acoustic artifacts, making it difficult to interpret auditory responses from recorded neurons. Here, we report the development of a silent two-photon imaging system and its applications in the common marmoset (Callithrix Jacchus), a non-human primate species sharing a similar hearing range with humans. By utilizing an orthogonal pair of acousto-optical deflectors (AODs), full-frame raster scanning at video rate was achieved without introducing mechanical vibrations. Imaging depth can be optically controlled by adjusting the chirping speed on the AODs without any mechanical motion along the Z-axis. Furthermore, all other sound-generating components of the system were acoustically isolated, leaving the noise floor of the working system below the marmoset’s hearing threshold. Imaging with the system in awake marmosets revealed many auditory cortex neurons that exhibited maximal responses at low sound levels, which were not possible to study using traditional two-photon imaging systems. This is the first demonstration of a silent two-photon imaging system that is capable of imaging auditory neuronal functions in vivo without acoustic artifacts. This capacity opens new opportunities for a better understanding of auditory functions in the brain and helps isolate animal behavior from microscope-generated acoustic interference.

Properties and fate of human mesenchymal stem cells upon miRNA let-7f-promoted recruitment to atherosclerotic plaques.

Atherosclerosis is a chronic inflammatory disease of the arteries leading to the formation of atheromatous plaques. Human mesenchymal stem cells (hMSCs) are recruited from the circulation into plaques where in response to their environment they adopt a phenotype with immunomodulatory properties. However, the mechanisms underlying hMSC function in these processes are unclear. Recently, we described that miRNA let-7f controls hMSC invasion guided by inflammatory cytokines and chemokines. Here, we investigated the role of let-7f in hMSC tropism to human atheromas and the effects of the plaque microenvironment on cell fate and release of soluble factors. Incubation of hMSCs with LL-37, an antimicrobial peptide abundantly found in plaques, increased biosynthesis of let-7f and N-formyl peptide receptor 2 (FPR2), enabling chemotactic invasion of the cells towards LL-37, as determined by qRT-PCR, flow cytometry, and cell invasion assay analysis. In an Apoe-/- mouse model of atherosclerosis, circulating hMSCs preferentially adhered to athero-prone endothelium. This property was facilitated by elevated levels of let-7f in the hMSCs, as assayed by ex vivo artery perfusion and two-photon laser scanning microscopy. Exposure of hMSCs to homogenized human atheromatous plaque material considerably induced the production of various cytokines, chemokines, matrix metalloproteinases, and tissue inhibitors of metalloproteinases, as studied by PCR array and western blot analysis. Moreover, exposure to human plaque extracts elicited differentiation of hMSCs into cells of the myogenic lineage, suggesting a potentially plaque-stabilizing effect. Our findings indicate that let-7f promotes hMSC tropism towards atheromas through the LL-37/FPR2 axis and demonstrate that hMSCs upon contact with human plaque environment develop a potentially athero-protective signature impacting the pathophysiology of atherosclerosis.

Improved Quantification of Cell Density in the Arterial Wall-A Novel Nucleus Splitting Approach Applied to 3D Two-Photon Laser-Scanning Microscopy.

Accurate information on vascular smooth muscle cell (VSMC) content, orientation, and distribution in blood vessels is indispensable to increase understanding of arterial remodeling and to improve modeling of vascular biomechanics. We have previously proposed an analysis method to automatically characterize VSMC orientation and transmural distribution in murine carotid arteries under well-controlled biomechanical conditions. However, coincident nuclei, erroneously detected as one large nucleus, were excluded from the analysis, hampering accurate VSMC content characterization and distorting transmural distributions. In the present study, therefore, we aim to (1) improve the previous method by adding a “nucleus splitting” procedure to split coinciding nuclei, (2) evaluate the accuracy of this novel method, and (3) test this method in a mouse model of VSMC apoptosis. After euthanasia, carotid arteries from SM22α-hDTR Apoe–/– and control Apoe–/– mice were bluntly dissected, excised, mounted in a biaxial biomechanical tester and brought to in vivo axial stretch and a pressure of 100 mmHg. Nuclei and elastin fibers were then stained using Syto-41 and Eosin-Y, respectively, and imaged using 3D two-photon laser scanning microscopy. Nuclei were segmented from images and coincident nuclei were split. The nucleus splitting procedure determines the likelihood that voxel pairs within coincident nuclei belong to the same nucleus and utilizes these likelihoods to identify individual nuclei using spectral clustering. Manual nucleus counts were used as a reference to assess the performance of our splitting procedure. Before and after splitting, automatic nucleus counts differed −26.6 ± 9.90% (p < 0.001) and −1.44 ± 7.05% (p = 0.467) from the manual reference, respectively. Whereas the slope of the relative difference between the manual and automated counts as a function of the manual count was significantly negative before splitting (p = 0.008), this slope became insignificant after splitting (p = 0.653). Smooth muscle apoptosis led to a 33.7% decrease in VSMC density (p = 0.008). Nucleus splitting improves the accuracy of automated cell content quantification in murine carotid arteries and overcomes the progressively worsening problem of coincident nuclei with increasing cell content in vessels. The presented image analysis framework provides a robust tool to quantify cell content, orientation, shape, and distribution in vessels to inform experimental and advanced computational studies on vascular structure and function.

Imaging Synaptic Glutamate Release with Two-Photon Microscopy in Organotypic Slice Cultures.

The strength of an excitatory synapse relies on the amount of glutamate it releases and on the amount of postsynaptic receptors responding to the released glutamate. Here we describe a strategy to investigate presynaptic release independently of postsynaptic receptors, using a genetically encoded glutamate indicator (GEGI) such as iGluSnFR to measure synaptic transmission in rodent organotypic slice cultures. We express the iGluSnFR in CA3 pyramidal cells and perform two-photon glutamate imaging on individual Schaffer collateral boutons in CA1. Sparse labeling is achieved via transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser scanning microscopy. A spiral scan path over an individual presynaptic bouton allows to sample at high temporal resolution the local release site in order to capture the peak of iGluSnFR transients.

Involvement of Endothelial Nitric Oxide Synthase in Cerebral Microcirculation and Oxygenation in Traumatic Brain Injury.

Traumatic brain injury (TBI) leads to cerebral microvascular dysfunction and cerebral ischemia. Endothelial nitric oxide synthase (eNOS) is a key regulator of vascular homeostasis. We aimed to assess the role of eNOS in cerebral blood flow (CBF) changes after TBI. Moderate TBI was induced in eNOS knockout (KO) and wild-type (WT) mice (8 per group). Cerebral microvascular tone, microvascular CBF (mCBF) and tissue oxygenation (NADH) were measured by two-photon laser scanning microscopy (2PLSM) before and 1h, 1day and 3days after TBI. Cerebrovascular reactivity (CVR) was evaluated by the hypercapnia test. Laser Doppler cortical flux (cLDF) was simultaneously measured in the perilesional area. One hr after TBI, cLDF was 59.4±8.2% and 60.3±9.1% from the baseline (p<0.05) in WT and eNOSKO, respectively. 2PLSM showed decreased arteriolar diameter, the number of functioning capillaries, mCBF and tissue oxygenation (p<0.05). At 1day, cLDF increased to 65.2±6.4% in the WT group, while it decreased to 56.1±7.2% in the eNOSKO mice. 2PLSM revealed a further decrease in the number of functioning capillaries, mCBF, and oxygen supply which was slightly milder in WT mice (p<0.05 from the baseline). On the third day after TBI, cLDF increased to 72±5.2% in the WT, while it stayed the same in the eNOSKO group (55.9±6.4%, p<0.05 from the WT). 2PLSM showed reduction in arterioles with vasospasm, increase in the number of functioning capillaries, and improvement in mCBF and tissue oxygen supply in WT, while no significant changes were observed in eNOS KO (p<0.05). CVR was impaired in both groups 1h after TBI, and improved by the third day in the WT, while staying impaired in eNOS KO. In the subacute TBI period, the significance of eNOS in maintaining cerebral microcirculation and oxygen supply increases with time after the injury.

Drag-Reducing Polymers Improve Vascular Hemodynamics and Tissue Oxygen Supply in Mouse Model of Diabetes Mellitus.

Diabetes mellitus (DM) is a chronic metabolic disease characterised by hyperglycaemia and glucose intolerance caused by impaired insulin action and/or defective insulin secretion. Long-term hyperglycaemia leads to various structural and functional microvascular changes within multiple tissues, including the brain, which involves blood-brain barrier alteration, inflammation and neuronal dysfunction. We have shown previously that drag-reducing polymers (DRP) improve microcirculation and tissue oxygen supply, thereby reducing neurologic impairment in different rat models of brain injury. We hypothesised that DRP could improve cerebral and skin microcirculation in the situation of progressive microangiopathies associated with diabetes using a mouse model of diabetes mellitus. Diabetes was induced in C57BL/6 J mice with five daily consecutive intraperitoneal injections of streptozotocin (50mg/kg/day). Animals with plasma glucose concentrations greater than 250mg/dL were considered diabetic and were used in the study following four months of diabetes. DRP (2ppm) was injected biweekly during the last two weeks of the experiment. Cortical and skin (ear) microvascular cerebral blood flow (mCBF) and tissue oxygen supply (NADH) were measured by two-photon laser scanning microscopy (2PLSM). Cerebrovascular reactivity (CVR) was evaluated by measuring changes in arteriolar diameters and NADH (tissue oxygen supply) during the hypercapnia test. Transient hypercapnia was induced by a 60-second increase of CO2 concentration in the inhalation mixture from 0% to 10%. Compared to non-diabetic animals, diabetic mice had a significant reduction in the density of functioning capillaries per mm3 (787±52 vs. 449±25), the linear velocity of blood flow (1.2±0.31 vs. 0.54±0.21mm/sec), and the tissue oxygen supply (p<0.05) in both brain and skin. DRP treatment was associated with a 50% increase in all three parameters (p<0.05). According to the hypercapnia test, CVR was impaired in both diabetic groups but more preserved in DRP mice (p<0.05). Our study in a diabetic mouse model has demonstrated the efficacy of hemorheological modulation of blood flow by DRP to achieve increased microcirculatory flows and tissue oxygen supply.

Halogen-modified carbazole derivatives for lipid droplet-specific bioimaging and two-photon photodynamic therapy

Lipid droplets (LDs) are dynamic multifunctional organelles that participate in the regulation of many metabolic processes, visualization of which is necessary for biological research. In this work, a series of two-photon responsive fluorescent probes (C-H, C-Br, and C-I) based on carbazole units were designed and synthesized. Thereinto, an iodine-modified carbazole derivative C-I exhibited an exciting lipid droplet targeting ability due to its excellent lipophilicity. Meanwhile, benefiting from its larger Stokes shift and two-photon absorption cross-section, C-I was employed for two-photon confocal laser scanning microscopy (CLSM) and stimulated emission depletion (STED) microscopy imaging to observe LDs more accurately. In addition, given the heavy atom effect, C-I can effectively generate reactive oxygen species (ROS) leading to cancer cell apoptosis under near-infrared light irradiation. Notably, we explained the process of cell apoptosis through in vitro simulation experiments. This study provides a promising platform for visualization of lipid droplets.