Two-photon Laser Scanning Microscopy Research Articles

An-Gong-Niu-Huang-Wan (AGNHW) regulates cerebral blood flow by improving hypoperfusion, cerebrovascular reactivity and microcirculation disturbances after stroke

BackgroundThe restoration of cerebrovascular regulation and improvement of cerebral blood flow in ischaemic regions are crucial for improving the clinical prognosis after stroke. An-Gong-Niu-Huang-Wan (AGNHW) is a famous traditional compound Chinese medicine that has been used for over 220 years to treat acute ischaemic stroke; however, its role in the regulation of cerebral blood flow is still unclear. The aim of the present study was to investigate the regulatory effect of AGNHW on cerebral blood flow and microcirculation after ischaemic stroke and to elucidate the underlying mechanisms involved.MethodsMale C57BL/6 mice were subjected to distal middle cerebral artery occlusion (dMCAO) and randomly assigned to the sham, MCAO, or AGNHW groups. AGNHW was administered intragastrically 1 h after dMCAO. The rotarod test was utilized to evaluate behavioural function; TTC was used to determine the infarct volume; and ischaemic injury was assessed by detecting brain levels of SOD, MDA and NO. Then, cortical perfusion and acetazolamide-induced cerebrovascular reactivity were assessed using laser speckle contrast imaging, and the velocity and flux of red blood cells in cortical capillaries were detected using two-photon laser scanning microscopy. In addition, we employed RNA-Seq to identify variations in gene expression profiles and assessed endothelium-dependent changes in microcirculatory dysfunction by measuring vasoactive mediator levels.ResultsAGNHW significantly increased cerebral blood flow, reduced the infarct volume, and promoted functional recovery after cerebral ischaemia. AGNHW increased the velocity and flux of red blood cells in capillaries and improved cerebrovascular reactivity in the ischaemic cortex. Furthermore, AGNHW regulated endothelium-dependent microcirculation, as evidenced by decreases in the expression of endothelins (Edn1, Edn3 and Ednrb) and the ratios of brain and serum TXB2/6-keto-PGF1α and ET-1/CGRP.ConclusionsAGNHW improved cerebral hypoperfusion, regulated cerebrovascular reactivity and attenuated microcirculatory dysfunction within the ischaemic cortex after stroke. This outstanding effect was achieved by modulating the expression of genes related to vascular endothelial cell function and regulating endothelium-dependent vasoactive mediators.

Contractile pericyte-mediated cerebrovascular deficits in a mouse model of CADASIL

Small vessel disease (SVD) of the brain underlies 25% of stroke cases and is the most common cause of vascular cognitive impairment. The most common monogenic form of SVD is CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), caused by mutations of the NOTCH3 protein which is widely expressed in mural cells (vascular smooth muscle cells [SMCs] and pericytes). Cerebral blood flow (CBF) deficits present early in CADASIL, attributable in part to NOTCH3-mediated extracellular matrix dysregulation, which alters heparin-binding epidermal growth factor-like (HB-EGF) signaling in SMCs and endothelial cells (ECs). Pericytes also robustly express NOTCH3 and are a relatively underexplored cell type in the context of CADASIL. Pericytes proximal to the penetrating arteriole (PA), known as contractile pericytes, express α-smooth muscle actin (αSMA) and can contract to rapidly regulate capillary diameter and thus blood flow in the downstream capillary tree. We conducted two-photon laser scanning microscopy (2PLSM) of blood flow and pericyte contractile responses with CADASIL Notch3R169C transgenic and control mice. Our data indicates that contractile pericyte-mediated control of blood flow is impaired prior to other known CBF deficits, thus raising the tantalizing possibility that these pericytes could be a target for early clinical intervention in CADASIL. While PA responses to stimulation of capillary electrical signaling remain intact, both blood flow as well as capillary diameter of contractile pericyte-covered vessels are diminished. Taken together, this work will establish pericytes as a novel site of dysfunction and provide a new candidate mechanism by which CBF is diminished early in CADASIL pathogenesis for potential therapeutic intervention. 5T32GM008181-33 (AV), 1DP2OD02944801 (TL), 1R01AG066645 (TL), 5R01NS115401 (TL). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ca2+ oscillation in vascular smooth muscle cells control myogenic spontaneous vasomotion and counteract post-ischemic no-reflow

Ischemic stroke produces the highest adult disability. Despite successful recanalization, no-reflow, or the futile restoration of the cerebral perfusion after ischemia, is a major cause of brain lesion expansion. However, the vascular mechanism underlying this hypoperfusion is largely unknown, and no approach is available to actively promote optimal reperfusion to treat no-reflow. Here, by combining two-photon laser scanning microscopy (2PLSM) and a mouse middle cerebral arteriolar occlusion (MCAO) model, we find myogenic vasomotion deficits correlated with post-ischemic cerebral circulation interruptions and no-reflow. Transient occlusion-induced transient loss of mitochondrial membrane potential (ΔΨm) permanently impairs mitochondria-endoplasmic reticulum (ER) contacts and abolish Ca2+ oscillation in smooth muscle cells (SMCs), the driving force of myogenic spontaneous vasomotion. Furthermore, tethering mitochondria and ER by specific overexpression of ME-Linker in SMCs restores cytosolic Ca2+ homeostasis, remotivates myogenic spontaneous vasomotion, achieves optimal reperfusion, and ameliorates neurological injury. Collectively, the maintaining of arteriolar myogenic vasomotion and mitochondria-ER contacts in SMCs, are of critical importance in preventing post-ischemic no-reflow.

Astrogliosis: A review of the astrocytic mechanisms, imaging modes, and treatments in spinal cord injury patients

Astrocytes are cells in the central nervous system (CNS) that are responsible for many things, such as maintaining blood brain barrier (BBB), regulating synapses in the spinal cord, and responding to spinal cord injury (SCI). Astrogliosis, the astrocytic response to spinal cord injuries (SCIs), helps repair CNS damages by regulating different protein filaments, thus limiting axonal growth. Former studies that were demonstrated through laser capture microdissection and immunohistochemistry (IHC) helped to identify important genes involved in experimental therapies for SCIs. Additionally, there are potential clinical treatments options for SCIs such as hydrogels, mesenchymal stem cells and steroids. Increased imaging modalities indicate that excessive astrogliosis can have adverse effects. These imaging techniques include positron emission tomography (PET), magnetic resonance imaging (MRI), and two-photon laser-scanning microscopy (TPLSM). These techniques illuminate greater details of the astrocytic response to SCIs. Despite these findings, astrogliosis is not well understood by the research community. Many of the studies presented in this literature review are experimental attempts to understand the mechanisms of astrogliosis in SCIs. This literature review aims to summarize the methods of each study in visualizing the mechanisms of astrogliosis and how they play a role in SCIs. Furthermore, this paper is aimed to comprehensively bridge the developments in the treatment for SCI patients based on innovative imaging modalities. Compared to prior studies, this review utilizes more recent understandings of the astrogliosis mechanisms to highlight insights into targeted developments, both clinically and preclinically. Some limitations of this literature review include the limited studies on astrogliosis and its impact on SCIs. Nonetheless, there is ongoing potential in the search for treatments for SCIs.

Monitoring the Intracellular pH and Metabolic State of Cancer Cells in Response to Chemotherapy Using a Combination of Phosphorescence Lifetime Imaging Microscopy and Fluorescence Lifetime Imaging Microscopy.

The extracellular matrix (ECM), in which collagen is the most abundant protein, impacts many aspects of tumor physiology, including cellular metabolism and intracellular pH (pHi), as well as the efficacy of chemotherapy. Meanwhile, the role of collagen in differential cell responses to treatment within heterogeneous tumor environments remains poorly investigated. In the present study, we simultaneously monitored the changes in pHi and metabolism in living colorectal cancer cells in vitro upon treatment with a chemotherapeutic combination, FOLFOX (5-fluorouracil, oxaliplatin and leucovorin). The pHi was followed using the new pH-sensitive probe BC-Ga-Ir, working in the mode of phosphorescence lifetime imaging (PLIM), and metabolism was assessed from the autofluorescence of the metabolic cofactor NAD(P)H using fluorescence lifetime imaging (FLIM) with a two-photon laser scanning microscope. To model the ECM, 3D collagen-based hydrogels were used, and comparisons with conventional monolayer cells were made. It was found that FOLFOX treatment caused an early temporal intracellular acidification (reduction in pHi), followed by a shift to more alkaline values, and changed cellular metabolism to a more oxidative state. The presence of unstructured collagen markedly reduced the cytotoxic effects of FOLFOX, and delayed and diminished the pHi and metabolic responses. These results support the observation that collagen is a factor in the heterogeneous response of cancer cells to chemotherapy and a powerful regulator of their metabolic behavior.

EXTH-73. IN VIVO EFFECTS OF EPCAM-DIRECTED CAR T CELLS IN COMBINATION WITH PD-1 CHECKPOINT BLOCKADE IN LUNG CANCER BRAIN METASTASIS

Abstract BACKGROUND Metastatic lung cancer is incurable once it spreads to the brain. Chimeric antigen receptor (CAR) T cells show promise in hematologic malignancies but face challenges in solid tumors, particularly in brain tumors, due to a highly immunosuppressive tumor microenvironment (TME), tumor antigen (TA) heterogeneity and impeded infiltration and persistence at the tumor site. To test the efficacy of CAR T cells and to find strategies for potentially enhancing its efficacy, CAR T cells were applied with and without checkpoint-blockade to treat lung cancer brain metastasis. METHODS We used an immunocompetent syngeneic orthotopic cerebral metastasis model combined with a chronic cranial window and repetitive intracerebral two-photon laser scanning microscopy (TPLSM). This allowed us to observe fluorescent tumor cells and CAR T cells in vivo at a single cell level over time. We injected red fluorescent EpCAM-transduced Lewis Lung carcinoma cells (EpCAM-LL/2tdT) into the brain tissue, followed by injection of EpCAM-directed or mock CAR T cells. Animals received anti-PD-1 and the respective isotype control via intraperitoneal injections. RESULTS EpCAM-directed CAR T cells were generated and showed substantial anti-tumor effects in vitro. Local injection into EpCAM-LL/2 tumor-bearing mice led to intratumoral accumulation of CAR T cells, resulting in reduced tumor growth compared to undirected CAR T cells translating into long-term survival in a fraction of animals. However, intratumoral CAR T cell numbers decrease over time pointing towards insufficient persistence. Interestingly, anti-PD-1 treatment did not enhance intratumoral CAR T accumulation, persistence and anti-tumor efficacy. CONCLUSION Our findings demonstrate the great potential of cell-based treatment approaches for the treatment of lung cancer brain metastases and underlines the necessity of further investigations to enhance infiltration and persistence in solid tumors.

EXTH-25. IN VIVO TWO-PHOTON TUMOR IMAGING IN AN ORTHOTOPIC MEDULLOBLASTOMA MOUSE MODEL

Abstract BACKGROUND Medulloblastoma represents the most common primary brain malignancy in pediatric patients and is associated with neuro-cognitive impairment and frequent relapses after aggressive multimodal therapy. Adoptive immunotherapies such as CAR T-cell therapy are currently being investigated, showing promising results in preclinical models but lacking efficacy in first-in-human trials. Refined in vivo models to investigate reasons for treatment failures are therefore urgently needed. METHODS We developed a xenogeneic orthotopic medulloblastoma (MB) model in mice by combining a chronic cerebellar window with repetitive intravital two-photon laser scanning microscopy. Red tdTomato-fluorescent DAOY (SHH MB) tumor cells expressing B7-H3 were implanted intracranially into the cerebellum of immunodeficient mice (n = 7), and tumor cell formation was followed by in vivo-microscopy. Intravenous fluorescein isothiocyanate (FITC)-dextran was used for intravascular plasma staining. B7-H3-directed CAR T-cells were injected into the adjacent brain parenchyma. RESULTS Chronic cranial window implantation was well tolerated and allowed repetitive visualization of the mouse cerebellum. After intracranial tumor cell implantation, continuous tumor growth could be evaluated with epifluorescence as well as 2-photon laser scanning microscopy. Tumor formation was identified as early as day 5 in every mouse and could continuously be followed up to 35 days until mice succumbed due to tumor burden. Window quality and fluorescence intensity remained high until the end of the experiments. Studies on GFP-expressing CAR T-cell injection directed against B7-H3 are currently ongoing. CONCLUSIONS We herein establish a robust orthotopic medulloblastoma mouse model that allows repetitive in vivo tracking of fluorescence-expressing tumor cells, CAR T-cells and blood vessels over weeks. Such models may be used to study medulloblastoma tumor growth under the influence of different immunotherapies and reveal interactions between tumor cells and CAR T-cell therapies on a single-cell level.

OS09.1.AIN VIVOTWO-PHOTON TUMOR IMAGING IN AN ORTHOTOPIC MEDULLOBLASTOMA MOUSE MODEL

Abstract BACKGROUND Medulloblastoma represents the most common primary brain malignancy in pediatric patients and is associated with neuro-cognitive impairment and frequent relapses after aggressive multimodal therapy. Adoptive immunotherapies such as CAR T-cell therapy are currently being investigated, showing promising results in preclinical models but lacking efficacy in first-in-human trials. Refined in vivo models to investigate reasons for treatment failures are therefore urgently needed. METHODS We developed a xenogeneic orthotopic medulloblastoma (MB) model in mice by combining a chronic cerebellar window with repetitive intravital two-photon laser scanning microscopy. Red tdTomato-fluorescent DAOY (SHH MB) tumor cells expressing EphA2 were implanted intracranially into the cerebellum of immunodeficient mice (n = 7), and tumor cell formation was followed by in vivo-microscopy. Intravenous fluorescein isothiocyanate (FITC)-dextran was used for intravascular plasma staining. EphA-directed CAR T-cells were injected into the adjacent brain parenchyma. RESULTS Chronic cranial window implantation was well tolerated and allowed repetitive visualization of the mouse cerebellum. After intracranial tumor cell implantation, continuous tumor growth could be evaluated with epifluorescence as well as 2-photon laser scanning microscopy. Tumor formation was identified as early as day 5 in every mouse and could continuously be followed up to 35 days until mice succumbed due to tumor burden. Window quality and fluorescence intensity remained high until the end of the experiments. Studies on GFP-expressing CAR T-cell injection directed against EphA2 are currently ongoing. CONCLUSIONS We herein establish a robust orthotopic medulloblastoma mouse model that allows repetitive in vivo tracking of fluorescence-expressing tumor cells, CAR T-cells and blood vessels over weeks. Such models may be used to study medulloblastoma tumor growth under the influence of different immunotherapies and reveal interactions between tumor cells and CAR T-cell therapies on a single-cell level.

Impact of the Protein Environment on Two-Photon Absorption Cross-Sections of the GFP Chromophore Anion Resolved at the XMCQDPT2 Level of Theory.

The search for fluorescent proteins with large two-photon absorption (TPA) cross-sections and improved brightness is required for their efficient use in bioimaging. Here, we explored the impact of a single-point mutation close to the anionic form of the GFP chromophore on its TPA activity. We considered the lowest-energy transition of EGFP and its modification EGFP T203I. We focused on a methodology for obtaining reliable TPA cross-sections for mutated proteins, based on conformational sampling using molecular dynamics simulations and a high-level XMCQDPT2-based QM/MM approach. We also studied the numerical convergence of the sum-over-states formalism and provide direct evidence for the applicability of the two-level model for calculating TPA cross-sections in EGFP. The calculated values were found to be very sensitive to changes in the permanent dipole moments between the ground and excited states and highly tunable by internal electric field of the protein environment. In the case of the GFP chromophore anion, even a single hydrogen bond was shown to be capable of drastically increasing the TPA cross-section. Such high tunability of the nonlinear photophysical properties of the chromophore anions can be used for the rational design of brighter fluorescent proteins for bioimaging using two-photon laser scanning microscopy.

In the mouse cortex, oligodendrocytes regain a plastic capacity, transforming into astrocytes after acute injury.

Acute brain injuries evoke various response cascades directing the formation of the glial scar. Here, we report that acute lesions associated with hemorrhagic injuries trigger a re-programming of oligodendrocytes. Single-cell RNA sequencing highlighted a subpopulation of oligodendrocytes activating astroglial genes after acute brain injuries. By using PLP-DsRed1/GFAP-EGFP and PLP-EGFPmem/GFAP-mRFP1 transgenic mice, we visualized this population of oligodendrocytes that we termed AO cells based on their concomitant activity of astro- and oligodendroglial genes. By fate mapping using PLP- and GFAP-split Cre complementation and repeated chronic invivo imaging with two-photon laser-scanning microscopy, we observed the conversion of oligodendrocytes into astrocytes via the AO cell stage. Such conversion was promoted by local injection of IL-6 and was diminished by IL-6 receptor-neutralizing antibody as well as by inhibiting microglial activation with minocycline. In summary, our findings highlight the plastic potential of oligodendrocytes in acute brain trauma due to microglia-derived IL-6.

Modified mouse model of repeated mild traumatic brain injury through a thinned-skull window and fluid percussion.

Mild traumatic brain injury (mTBI) is a clinically highly heterogeneous neurological disorder, none of the existing animal models can replicate the entire sequelae. This study aimed to develop a modified closed head injury (CHI) model of repeated mTBI (rmTBI) for investigating Ca2+ fluctuations of the affected neural network, the alternations of electrophysiology, and behavioral dysfunctions. The transcranial Ca2+ study protocol includes AAV-GCaMP6s infection in the right motor cortex, thinned-skull preparation, and two-photon laser scanning microscopy (TPLSM) imaging. The CHI rmTBI model is fabricated using the thinned-skull site and applying 2.0 atm fluid percussion with 48-h interval. The neurological dysfunction, minor motor performance, evident mood, spatial working, and reference deficits we found in this study mimic the clinically relevant syndromes after mTBI. Besides, our study revealed that there was a trend of transition from Ca2+ singlepeak to multipeak and plateau, and the total Ca2+ activities of multipeaks and plateaus (p < .001 vs. pre-rmTBI value) were significantly increased in ipsilateral layer 2/3 motor neurons after rm TBI. In parallel, there is a low-frequency power shift from delta to theta band (p < .01 vs. control) in the ipsilateral layer 2/3 of motor cortex of the rmTBI mice, and the overall firing rates significantly increased (p < .01 vs. control). Moreover, rmTBI causes slight cortical and hippocampal neuron damage and possibly induces neurogenesis in the dentate gyrus (DG). The alternations of Ca2+ and electrophysiological characteristics in layer 2/3 neuronal network, histopathological changes, and possible neurogenesis may play concertedly and partially contribute to the functional outcome post-rmTBI.

Individual glioblastoma cells harbor both proliferative and invasive capabilities during tumor progression.

Glioblastomas are characterized by aggressive and infiltrative growth, and by striking heterogeneity. The aim of this study was to investigate whether tumor cell proliferation and invasion are interrelated, or rather distinct features of different cell populations. Tumor cell invasion and proliferation were longitudinally determined in real-time using 3D in vivo 2-photon laser scanning microscopy over weeks. Glioblastoma cells expressed fluorescent markers that permitted the identification of their mitotic history or their cycling versus non-cycling cell state. Live reporter systems were established that allowed us to dynamically determine the invasive behavior, and previous or actual proliferation of distinct glioblastoma cells, in different tumor regions and disease stages over time. Particularly invasive tumor cells that migrated far away from the main tumor mass, when followed over weeks, had a history of marked proliferation and maintained their proliferative capacity during brain colonization. Infiltrating cells showed fewer connections to the multicellular tumor cell network, a typical feature of gliomas. Once tumor cells colonized a new brain region, their phenotype progressively transitioned into tumor microtube-rich, interconnected, slower-cycling glioblastoma cells. Analysis of resected human glioblastomas confirmed a higher proliferative potential of tumor cells from the invasion zone. The detection of glioblastoma cells that harbor both particularly high proliferative and invasive capabilities during brain tumor progression provides valuable insights into the interrelatedness of proliferation and migration-2 central traits of malignancy in glioma. This contributes to our understanding of how the brain is efficiently colonized in this disease.

The counterbalance of endothelial glycocalyx and high wall shear stress to low-density lipoprotein concentration polarization in mouse ear skin arterioles

Background and aimsAtherosclerosis preferentially occurs at regions in arterial branching, curvature, and stenosis, which may be explained by the geometric predilection of low-density lipoprotein (LDL) concentration polarization that has been investigated in major arteries in previous studies. Whether this also happens in arterioles remains unknown. MethodsHerein, a radially non-uniform distribution of LDL particles and a heterogeneous endothelial glycocalyx layer in the mouse ear arterioles, as shown by fluorescein isothiocyanate labeled wheat germ agglutinin (WGA-FITC), were successfully observed by a non-invasive two-photon laser-scanning microscopy (TPLSM) technique. The stagnant film theory was applied as the fitting function to evaluate LDL concentration polarization in arterioles. ResultsThe concentration polarization rate (CPR, the ratio of the number of polarized cases to that of total cases) in the inner walls of curved and branched arterioles was 22% and 31% higher than the outer counterparts, respectively. Results from the binary logistic regression and multiple linear regression analysis showed that endothelial glycocalyx thickness increases CPR and the thickness of the concentration polarization layer (CPL). Flow field computation indicates no obvious disturbances or vortex in modeled arterioles with different geometries and the mean wall shear stress is about 7.7–9.0 Pa. ConclusionsThese findings suggest a geometric predilection of LDL concentration polarization in arterioles for the first time, and the existence of an endothelial glycocalyx, acting together with a relatively high wall shear stress in arterioles, may explain to some extent why atherosclerosis rarely occurs in these regions.

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.