A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord,

M. J. Farrar and C. B. Schaffer

Journal of Visualized Experiments (2014)

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Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

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Constitutively active Notch4 receptor elicits brain arteriovenous malformations through enlargement of capillary-like vessels

Patrick A. Murphya, Tyson N. Kima, Lawrence Huanga, Corinne M. Nielsena , Michael T. Lawtonb , Ralf H. Adamsc , Chris B. Schafferd , and Rong A. Wanga,

Proceedings of the National Academy of Sciences (2014)

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Arteriovenous (AV) malformation (AVM) is a devastating condition characterized by focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. AVMs can form anywhere in the body and can cause debilitating ischemia and life-threatening hemorrhagic stroke. The mechanisms that underlie AVM formation remain poorly understood. Here, we examined the cellular and hemodynamic changes at the earliest stages of brain AVM formation by time-lapse two-photon imaging through cranial windows of mice expressing constitutively active Notch4 (Notch4*). AVMs arose from enlargement of preexisting microvessels with capillary diameter and blood flow and no smooth muscle cell coverage. AV shunting began promptly after Notch4* expression in endothelial cells (ECs), accompanied by increased individual EC areas, rather than increased EC number or proliferation. Alterations in Notch signaling in ECs of all vessels, but not arteries alone, affected AVM formation, suggesting that Notch functions in the microvasculature and/or veins to induce AVM. Increased Notch signaling interfered with the normal biological control of hemodynamics, permitting a positive feedback loop of increasing blood flow and vessel diameter and driving focal AVM growth from AV connections with higher blood velocity at the expense of adjacent AV connections with lower velocity. Endothelial expression of constitutively active Notch1 also led to brain AVMs in mice. Our data shed light on cellular and hemodynamic mechanisms underlying AVM pathogenesis elicited by increased Notch signaling in the endothelium.

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Mechanistic insight into the TH1-biased immune response to recombinant subunit vaccines delivered by probiotic bacteria-derived outer membrane vesicles

Rosenthal JA, Huang CJ, Doody AM, Leung T, Mineta K, Feng DD, Wayne EC, Nishimura N, Leifer C, DeLisa MP, Mendez S, Putnam D.

PLoS One. (2014)

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Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV's ability to induce self-adjuvanted robust and protective humoral and T(H)1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered "pathogen-like particles" derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion.

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Stalled cerebral capillary blood flow in mouse models of essential thrombocythemia and polycythemia vera revealed by in vivo two-photon imaging.

Santisakultarm TP, Paduano CQ, Stokol T, Southard TL, Nishimura N, Skoda RC, Olbricht WL, Schafer AI, Silver RT, Schaffer CB.

J Thromb Haemost. (2014)

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Background Essential thrombocythemia (ET) and polycythemia vera (PV) are myeloproliferative neoplasms (MPNs) that share the JAK2V617F mutation in hematopoietic stem cells, leading to excessive production of predominantly platelets in ET, and predominantly red blood cells (RBCs) in PV. The major cause of morbidity and mortality in PV and ET is thrombosis, including cerebrovascular occlusive disease. Objectives To identify the effect of excessive blood cells on cerebral microcirculation in ET and PV. Methods We used two-photon excited fluorescence microscopy to examine cerebral blood flow in transgenic mouse models that mimic MPNs. Results and conclusions We found that flow was ‘stalled’ in an elevated fraction of brain capillaries in ET (18%), PV (27%), mixed MPN (14%) and secondary (non-MPN) erythrocytosis (27%) mice, as compared with controls (3%). The fraction of capillaries with stalled flow increased when the hematocrit value exceeded 55% in PV mice, and the majority of stalled vessels contained only stationary RBCs. In contrast, the majority of stalls in ET mice were caused by platelet aggregates. Stalls had a median persistence time of 0.5 and 1 h in ET and PV mice, respectively. Our findings shed new light on potential mechanisms of neurological problems in patients with MPNs.

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TRAIL-coated leukocytes that kill cancer cells in the circulation

Michael J. Mitchell, Elizabeth Wayne, Kuldeepsinh Rana, Chris B. Schaffer, and Michael R. King

Proceedings of the National Academy of Sciences USA (2014)

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Metastasis through the bloodstream contributes to poor prognosis in many types of cancer. Mounting evidence implicates selectin- based adhesive interactions between cancer cells and the blood vessel wall as facilitating this process, in a manner similar to leukocyte trafficking during inflammation. Here, we describe a unique approach to target and kill colon and prostate cancer cells in the blood that causes circulating leukocytes to present the cancer-specific TNF-related apoptosis inducing ligand (TRAIL) on their surface along with E-selectin adhesion receptor. This approach, demonstrated in vitro with human blood and also in mice, mimics the cytotoxic activity of natural killer cells and increases the sur- face area available for delivery of the receptor-mediated signal. The resulting “unnatural killer cells” hold promise as an effective means to neutralize circulating tumor cells that enter blood with the potential to form new metastases.

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