Choosing a polarisation configuration for dynamic light scattering and laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) is applied in various biomedical applications for full-field characterization of blood flow and tissue perfusion. The accuracy of the contrast interpretation and its conversion to the blood flow index depends on specific parameters of the optical system and scattering media. One such parameter is the polarisation of detected light, which is often adjusted to minimize specular reflections and image artefacts. The polarisation's effect on the detected light scattering dynamics and, therefore, the accuracy of LSCI data interpretation requires more detailed investigation. In this study, we used LSCI and Dynamic Light Scattering Imaging to evaluate the effects of the detected light polarisation when imaging perfusion in the mouse cortex. We found that cross-polarisation results in a shorter decorrelation time constant, a higher coherence degree and stronger dynamic scattering compared to the parallel-polarisation or no-polariser configurations. These results support the cross-polarisation configuration as the most optimal for brain cortex imaging and suggest against direct or calibrated comparisons between the contrast recordings made with different polarisation configurations.

Liver-secreted fluorescent blood plasma markers enable chronic imaging of the microcirculation.

Studying blood microcirculation is vital for gaining insights into vascular diseases. Blood flow imaging in deep tissue is currently achieved by acute administration of fluorescent dyes in the blood plasma. This is an invasive process, and the plasma fluorescence decreases within an hour of administration. Here, we report an approach for the longitudinal study of vasculature. Using a single intraperitoneal or intravenous administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to chronically label the blood circulation in mice. This approach allows for longitudinal observation of circulation from 2 weeks to over 4 months after vector administration. We demonstrate the chronic assessment of vascular functions including functional hyperemia and vascular plasticity in micro- and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease animal models.

Assessment of astrocytes as a mediator of memory and learning in rodents.

The classical view of astrocytes is that they provide supportive functions for neurons, transporting metabolites and maintaining the homeostasis of the extracellular milieu. This view is gradually changing with the advent of molecular genetics and optical methods allowing interrogation of selected cell types in live experimental animals. An emerging view that astrocytes additionally act as a mediator of synaptic plasticity and contribute to learning processes has gained in vitro and in vivo experimental support. Here we focus on the literature published in the past two decades to review the roles of astrocytes in brain plasticity in rodents, whereby the roles of neurotransmitters and neuromodulators are considered to be comparable to those in humans. We outline established inputs and outputs of astrocytes and discuss how manipulations of astrocytes have impacted the behavior in various learning paradigms. Multiple studies suggest that the contribution of astrocytes has a considerably longer time course than neuronal activation, indicating metabolic roles of astrocytes. We advocate that exploring upstream and downstream mechanisms of astrocytic activation will further provide insight into brain plasticity and memory/learning impairment.

Transient Astrocytic Gq Signaling Underlies Remote Memory Enhancement.

Astrocytes elicit transient Ca2+ elevations induced by G protein-coupled receptors (GPCRs), yet their role in vivo remains unknown. To address this, transgenic mice with astrocytic expression of the optogenetic Gq-type GPCR, Optoα1AR, were established, in which transient Ca2+ elevations similar to those in wild type mice were induced by brief blue light illumination. Activation of cortical astrocytes resulted in an adenosine A1 receptor-dependent inhibition of neuronal activity. Moreover, sensory stimulation with astrocytic activation induced long-term depression of sensory evoked response. At the behavioral level, repeated astrocytic activation in the anterior cortex gradually affected novel open field exploratory behavior, and remote memory was enhanced in a novel object recognition task. These effects were blocked by A1 receptor antagonism. Together, we demonstrate that GPCR-triggered Ca2+ elevation in cortical astrocytes has causal impacts on neuronal activity and behavior.

Chemotherapeutic resistance of head and neck squamous cell carcinoma is mediated by EpCAM induction driven by IL-6/p62 associated Nrf2-antioxidant pathway activation.

Overexpression of epithelial cell adhesion molecule (EpCAM) has been associated with chemotherapeutic resistance, leads to aggressive tumor behavior, and results in an adverse clinical outcome. The molecular mechanism by which EpCAM enrichment is linked to therapeutic resistance via Nrf2, a key regulator of antioxidant genes is unknown. We have investigated the link between EpCAM and the Nrf2 pathway in light of therapeutic resistance using head and neck squamous cell carcinoma (HNSCC) patient tumor samples and cell lines. We report that EpCAM was highly expressed in Nrf2-positive and HPV-negative HNSCC cells. In addition, cisplatin-resistant tumor cells consisted of a higher proportion of EpCAMhigh cells compared to the cisplatin sensitive counterpart. EpCAMhigh populations exhibited resistance to cisplatin, a higher efficiency in colony formation, sphere growth and invasion capacity, and demonstrated reduced reactive oxygen species (ROS) activity. Furthermore, Nrf2 expression was significantly higher in EpCAMhigh populations. Mechanistically, expression of Nrf2 and its target genes were most prominently observed in EpCAMhigh populations. Silencing of EpCAM expression resulted in the attenuation of expressions of Nrf2 and SOD1 concomitant with a reduction of Sox2 expression. On the other hand, silencing of Nrf2 expression rendered EpCAMhigh populations sensitive to cisplatin treatment accompanied by the inhibition of colony formation, sphere formation, and invasion efficiency and increased ROS activity. The molecular mechanistic link between EpCAM expression and activation of Nrf2 was found to be a concerted interaction of interleukin-6 (IL-6) and p62. Silencing of p62 expression in EpCAMhigh populations resulted in the attenuation of Nrf2 pathway activation suggesting that Nrf2 pathway activation promoted resistance to cisplatin in EpCAMhigh populations. We propose that therapeutic targeting the Nrf2-EpCAM axis might be an excellent approach to modulate stress resistance and thereby survival of HNSCC patients enriched in EpCAMhigh populations.

Widespread expression of Sonic hedgehog (Shh) and Nrf2 in patients treated with cisplatin predicts outcome in resected tumors and are potential therapeutic targets for HPV-negative head and neck cancer.

BACKGROUND: Sonic hedgehog (Shh) and Nrf2 play a critical role in chemotherapeutic resistance. These two genes have been found to be dysregulated in head and neck squamous cell carcinomas (HNSCC). The purpose of this study was to analyze the expression, function and clinical prognostic relationship of Shh and Nrf2 in HNSCC in the context of therapeutic resistance and cancer stem cells (CSCs). METHODS: We analyzed a cohort of patients with HNSCC to identify potential therapeutic biomarkers correlating with overall survival (OS) as well as disease-free survival (DFS) from our own data and validated these results using The Cancer Genome Atlas dataset. Expression of Shh and Nrf2 was knocked down by siRNA and cell growth, sphere growth and chemotherapeutic resistance were evaluated. RESULTS: Widespread abundant expression of Shh and Nrf2 proteins were associated with shorter OS and DFS. The combination of Shh and Nrf2 expression levels was found to be a significant predictor of patient DFS. The tumor stromal index was correlated with Shh expression and inversely associated with shorter OS and DFS. Inhibition of Shh by siRNA or cyclopamine resulted in the attenuation of resistant CSC self-renewal, invasion, clonogenic growth and re-sensitization to the chemotherapeutic agents. Concomitant upregulation of Shh and Nrf2 proved to be an independent predictor of poor OS and DFS in patients with HNSCC. CONCLUSIONS: These findings suggest that Shh and Nrf2 could serve as therapeutic targets as well as promising dual prognostic therapeutic biomarkers for HNSCC.

Regional Distribution of Glycogen in the Mouse Brain Visualized by Immunohistochemistry.

Considering that the brain constantly consumes a substantial amount of energy, the nature of its energy reserve is an important issue. Although the brain is rich in lipid content encompassing membranes, myelin sheath, and astrocytic lipid droplets, it is devoid of adipose tissue which serves as an energy reserve. Notably, glycogen represents the major energy store in the brain. While glycogen has been observed mainly in astrocytes for decades by electron microscopy, glycogen distribution in the brain has only been partially documented. The involvement of glycogen metabolism in memory consolidation, demonstrated by several research groups, has reiterated the functional significance of this macromolecule and the need for description of its comprehensive distribution in the brain. The combination of focused microwave-assisted brain fixation and glycogen immunohistochemistry permits assessment of glycogen distribution in the rodent brain. In this article, we describe glycogen distribution in the mouse brain using glycogen immunohistochemistry. We find heterogeneous glycogen storage patterns at multiple spatial scales. The heterogeneous glycogen distribution patterns may underlie local energy metabolism or synaptic activity, and its mechanistic understanding should extend our knowledge on brain metabolism in health and disease.

Transcranial Direct Current Stimulation (tDCS) Induces Adrenergic Receptor-Dependent Microglial Morphological Changes in Mice.

Transcranial direct current stimulation (tDCS) has been reported for its beneficial effects on memory formation and various brain disorders. While the electrophysiological readout of tDCS effects is subtle, astrocytes have been demonstrated to elicit Ca2+ elevations during tDCS in a rodent model. This study aimed to elucidate the effects of tDCS on another major glial cell type, microglia, by histology and in vivo imaging. tDCS performed in awake conditions induced a significant change in the pixel intensity distribution of Iba-1 immunohistochemistry, and microglial somata were enlarged when examined 3 h after tDCS. These effects were blocked by adrenergic receptor antagonists or in IP3R2 (inositol trisphosphate receptor type 2)-deficient mice, which lack large cytosolic Ca2+ elevations in astrocytes. No obvious changes were observed in isoflurane-anesthetized mice. Furthermore, in vivo two-photon imaging of microglia showed a reduction of motility that was blocked by a ß2-adrenergic receptor antagonist. Our observations add support for the influence of noradrenaline in tDCS and suggest possible interactions between microglia and astrocytes to express functional changes associated with tDCS.

Glycogen distribution in mouse hippocampus.

The hippocampus is a limbic structure involved in the consolidation of episodic memory. In the recent decade, glycogenolysis in the rodent hippocampus has been shown critical for synaptic plasticity and memory formation. Astrocytes are the primary cells that store glycogen which is subject to degradation in hypoglycemic conditions. Focused microwave application to the brain halts metabolic activities, and therefore preserves brain glycogen. Immunohistochemistry against glycogen on focused microwave-assisted brain samples is suitable for both macroscopic and microscopic investigation of glycogen distribution. Glycogen immunohistochemistry in the hippocampus showed a characteristic punctate signal pattern that depended on hippocampal layers. In particular, the hilus is the most glycogen-rich subregion of the hippocampus. Moreover, large glycogen puncta (>0.5 µm in diameter) observed in neuropil areas are organized in a patchy pattern consisting of puncta-rich and -poor astrocytes. These observations are discussed with respect to distinct hippocampal neural activity states observed in live animals.