inducing-ischemia-reperfusion-injury-mouse-ear-skin-for-intravital

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Agency for Science, Technology and Research (A*STAR), Biopolis

National University of Singapore

Multiphoton (MP) microscopy enables the direct in vivo visualization, with high spatial and temporal resolution, of fluorescently tagged immune cells, extracellular matrix and vasculature in tissues. This approach, therefore, represents a powerful alternative to traditional methods of assessing immune cell function in the skin, which are mainly based on flow cytometry and histology. Here we provide a step-by-step protocol describing experimental procedures for intravital MP imaging of the mouse ear skin, which can be easily adapted to address many specific skin-related biological questions. We demonstrate the use of this procedure by characterizing the response of neutrophils during cutaneous inflammation, which can be used to perform in-depth analysis of neutrophil behavior in the context of the skin microanatomy, including the epidermis, dermis and blood vessels. Such experiments are typically completed within 1 d, but as the procedures are minimally invasive, it is possible to perform longitudinal studies through repeated imaging.

Intravital multiphoton imaging of immune responses in the mouse ear skin.

The cerebellum is a brain region responsible for motor coordination and for refining motor programs. While a great deal is known about the structure and connectivity of the mammalian cerebellum, fundamental questions regarding its function in behavior remain unanswered. Recently, the zebrafish has emerged as a useful model organism for cerebellar studies, owing in part to the similarity in cerebellar circuits between zebrafish and mammals. While the cell types composing their cerebellar cortical circuits are generally conserved with mammals, zebrafish lack deep cerebellar nuclei, and instead a majority of cerebellar output comes from a single type of neuron: the eurydendroid cell. To describe spatial patterns of cerebellar output in zebrafish, we have used genetic techniques to label and trace eurydendroid cells individually and en masse. We have found that cerebellar output targets the thalamus and optic tectum, and have confirmed the presence of pre-synaptic terminals from eurydendroid cells in these structures using a synaptically targeted GFP. By observing individual eurydendroid cells, we have shown that different medial-lateral regions of the cerebellum have eurydendroid cells projecting to different targets. Finally, we found topographic organization in the connectivity between the cerebellum and the optic tectum, where more medial eurydendroid cells project to the rostral tectum while lateral cells project to the caudal tectum. These findings indicate that there is spatial logic underpinning cerebellar output in zebrafish with likely implications for cerebellar function.

Cerebellar output in zebrafish: an analysis of spatial patterns and topography in eurydendroid cell projections.

Ultrabright organic dots with aggregation-induced emission characteristics (AIE dots) are prepared and shown to exhibit a high quantum yield, a, large two-photon absorption cross-section, and low in vivo toxicity. Real-time two-photon intravital blood vascular imaging in various tissues substantiates that the AIE dots are effective probes for in vivo vasculature imaging in a deep and high-contrast manner.

Ultrabright organic dots with aggregation-induced emission characteristics for real-time two-photon intravital vasculature imaging.

Blood neutrophil homeostasis is essential for successful host defense against invading pathogens. Circulating neutrophil counts are positively regulated by CXCR2 signaling and negatively regulated by the CXCR4-CXCL12 axis. In particular, G-CSF, a known CXCR2 signaler, and plerixafor, a CXCR4 antagonist, have both been shown to correct neutropenia in human patients. G-CSF directly induces neutrophil mobilization from the bone marrow (BM) into the blood, but the mechanisms underlying plerixafor-induced neutrophilia remain poorly defined. Using a combination of intravital multiphoton microscopy, genetically modified mice and novel in vivo homing assays, we demonstrate that G-CSF and plerixafor work through distinct mechanisms. In contrast to G-CSF, CXCR4 inhibition via plerixafor does not result in neutrophil mobilization from the BM. Instead, plerixafor augments the frequency of circulating neutrophils through their release from the marginated pool present in the lung, while simultaneously preventing neutrophil return to the BM. Our study demonstrates for the first time that drastic changes in blood neutrophils can originate from alternative reservoirs other than the BM, while implicating a role for CXCR4-CXCL12 interactions in regulating lung neutrophil margination. Collectively, our data provides valuable insights into the fundamental regulation of neutrophil homeostasis, which may lead to the development of improved treatment regimens for neutropenic patients.

Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow.

Basophils, a rare leukocyte population in peripheral circulation, are conventionally identified as CD45(int) CD49b(+) FcεRI(+) cells. Here, we show that basophils from blood and several organs of naïve wild-type mice express CD41, the α subunit of α(IIb)β₃ integrin. CD41 expression on basophils is upregulated after in vivo IL-3 treatment and during infection with Nippostrongylus brasiliensis (Nb). Moreover, CD41 can be used as a reliable marker for basophils, circumventing technical difficulties associated with FcεRI for basophil identification in a Nb infection model. In vitro anti-IgE cross-linking and IL-3 basophil stimulation showed that CD41 upregulation positively correlates with augmented surface expression of CD200R and increased production of IL-4/IL-13, indicating that CD41 is a basophil activation marker. Furthermore, we found that infection with Plasmodium yoelii 17X (Py17x) induced a profound basophilia and using Mcpt8(DTR) reporter mice as a basophil-specific depletion model, we verified that CD41 can be used as a marker to track basophils in the steady state and during infection. During malarial infection, CD41 expression on basophils is negatively regulated by IFN-γ and positively correlates with increased basophil IL-4 production. In conclusion, we provide evidence that CD41 can be used as both an identification and activation marker for basophils during homeostasis and immune challenge.

CD41 is a reliable identification and activation marker for murine basophils in the steady state and during helminth and malarial infections.

Real-time imaging of dendritic cell responses to sterile tissue injury.

Fluorescent organic dots are emerging as promising bioimaging reagents because of their high brightness, good photostability, excellent biocompatibility, and facile surface functionalization. Organic dots with large two-photon absorption (TPA) cross sections are highly desired for two-photon fluorescence microscopy. In this work, we report two biocompatible and photostable organic dots fabricated by encapsulating tetraphenylethene derivatives within DSPE-PEG matrix. The two organic dots show absorption maxima at 425 and 483 nm and emit green and red fluorescence at 560 and 645 nm, with high fluorescence quantum yields of 64% and 22%, respectively. Both organic dots exhibit excellent TPA property in the range of 800-960 nm, affording upon excitation at 820 nm remarkably large TPA cross sections of 1.2×10(6) and 2.5×10(6) GM on the basis of dot concentration. The bare fluorophores and their organic dots are biocompatible and have been used to stain living cells for one- and two-photon fluorescence bioimagings. The cRGD-modified organic dots can selectively target integrin αvβ3 overexpressing breast cancer cells for targeted imaging. The organic dots are also applied for real-time two-photon fluorescence in vivo visualization of the blood vasculature of mouse ear, providing the spatiotemporal information about the whole blood vascular network. These results demonstrate that the present fluorescent organic dots are promising candidates for living cell and tissue imaging.

Biocompatible Green and Red Fluorescent Organic Dots with Remarkably Large Two-Photon Action Cross Sections for Targeted Cellular Imaging and Real-Time Intravital Blood Vascular Visualization.

A polymer and silica co-protection strategy has been developed to encapsulate organic fluorogens with aggregation-induced emission and charge transfer characteristics into small nanoparticles (NPs). The co-pretected NPs show bright red fluorescence (50% quantum yield) with a large two-photon action cross-section (450 GM at 840 nm), which have been sucessfully used for two-photon fluorescence imaging of vasculature of the mouse tibial muscle.

Silica shelled and block copolymer encapsulated red-emissive AIE nanoparticles with 50% quantum yield for two-photon excited vascular imaging.

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation, and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine and/or paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.

Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation through CXCL2.

A novel theranostic divalent vancomycin analog using a planar 1,8-diazapyrene moiety as a rigid scaffold exhibits potent and selective antibacterial activity against Gram (+) bacteria including vancomycin-resistant strains, while having minimal influence on Gram (-) bacteria and mammalian cells. Moreover, this theranostic analog can be also applied for selective two-photon fluorescence imaging of Gram (+) bacteria.

Glycopeptide antibiotic analogs for selective inactivation and two-photon imaging of vancomycin-resistant strains.

A new bottom-up nanocrystallization method has been developed to fabricate highly fluorescent organic nanocrystals in aqueous media using an aggregation-induced emission fluorogen (AIEgen) as an example. The nanocrystallization strategy has led to the fabrication of uniform nanocrystals of 110 ± 10 nm size in aqueous media, which shows over 400% increase in brightness as compared to the amorphous nanoaggregates.

Article	Year
Intravital multiphoton imaging of immune responses in the mouse ear skin. Nature protocols\| PubMed ID: 22240584	2012
Cerebellar output in zebrafish: an analysis of spatial patterns and topography in eurydendroid cell projections. Frontiers in neural circuits\| PubMed ID: 23554587	2013
Ultrabright organic dots with aggregation-induced emission characteristics for real-time two-photon intravital vasculature imaging. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 24038281	2013
Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow. The Journal of experimental medicine\| PubMed ID: 24081949	2013
CD41 is a reliable identification and activation marker for murine basophils in the steady state and during helminth and malarial infections. European journal of immunology\| PubMed ID: 24610714	2014
Real-time imaging of dendritic cell responses to sterile tissue injury. The Journal of investigative dermatology\| PubMed ID: 25431854	2015
Biocompatible Green and Red Fluorescent Organic Dots with Remarkably Large Two-Photon Action Cross Sections for Targeted Cellular Imaging and Real-Time Intravital Blood Vascular Visualizatio... ACS applied materials & interfaces\| PubMed ID: 26094687	2015
Silica shelled and block copolymer encapsulated red-emissive AIE nanoparticles with 50% quantum yield for two-photon excited vascular imaging. Chemical communications (Cambridge, England)\| PubMed ID: 26213866	2015
Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation through CXCL2. The Journal of investigative dermatology\| PubMed ID: 26802238	2016
Glycopeptide antibiotic analogs for selective inactivation and two-photon imaging of vancomycin-resistant strains. Chemical communications (Cambridge, England)\| PubMed ID: 26953360	2016
Nanocrystallization: A Unique Approach to Yield Bright Organic Nanocrystals for Biological Applications. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 27805762	2016

Chi Ching Goh

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