scholarly journals Model System for Live Imaging of Neuronal Responses to Injury and Repair

2011 ◽  
Vol 10 (6) ◽  
pp. 7290.2011.00013 ◽  
Author(s):  
Mathieu Gravel ◽  
Yuan-Cheng Weng ◽  
Jasna Kriz
Keyword(s):  
Nervous System ◽  
Neurite Outgrowth ◽  
Fluorescence Imaging ◽  
Synapse Formation ◽  
Fluorescent Protein ◽  
Model System ◽  
Fluorescence Signal ◽  
Neuronal Responses ◽  
Injury And Repair

Although it has been well established that induction of growth-associated protein-43 (GAP-43) during development coincides with axonal outgrowth and early synapse formation, the existence of neuronal plasticity and neurite outgrowth in the adult central nervous system after injuries is more controversial. To visualize the processes of neuronal injury and repair in living animals, we generated reporter mice for bioluminescence and fluorescence imaging bearing the luc (luciferase) and gfp (green fluorescent protein) reporter genes under the control of the murine GAP-43 promoter. Reporter functionality was first observed during the development of transgenic embryos. Using in vivo bioluminescence and fluorescence imaging, we visualized induction of the GAP-43 signals from live embryos starting at E10.5, as well as neuronal responses to brain and peripheral nerve injuries (the signals peaked at 14 days postinjury). Moreover, three-dimensional analysis of the GAP-43 bioluminescent signal confirmed that it originated from brain structures affected by ischemic injury. The analysis of fluorescence signal at cellular level revealed colocalization between endogenous protein and the GAP-43-driven gfp transgene. Taken together, our results suggest that the GAP-43-luc/gfp reporter mouse represents a valid model system for real-time analysis of neurite outgrowth and the capacity of the adult nervous system to regenerate after injuries.

scholarly journals MMP-14-targeted Nanoprobe for in vivo Fluorescence Imaging of Rupture-prone Carotid Plaques

2021 ◽  
Author(s):  
Mengtao Han ◽  
Kaining Liu ◽  
Hongqiu Xiao ◽  
Tao Sun ◽  
Fei Wang ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Fluorescence Signal ◽  
Plaque Progression ◽  
Carotid Plaques ◽  
In Vivo Fluorescence ◽  
Cell Tissue ◽  
A Cell ◽  
In Vivo Fluorescence Imaging ◽  
Membrane Anchoring

Abstract Background: The identification of rupture-prone carotid plaques for preventing stroke remains a clinical challenge. Macrophage matrix metalloproteinase (MMP)-14, which contributes to plaque progression and destabilisation, could be a promising biomarker for plaque imaging. This study aimed to design and synthesise an MMP-14-targeted nanoprobe to noninvasively visualise the behaviour of M1 macrophages in atherosclerotic plaques.Methods: A fluorescence molecular imaging probe ([email protected]) was constructed by covalently attaching the fluorescent dye cyanine (Cy) 5.5, an MMP-14 substrate, and polyethylene glycol (PEG) 5000-wrapped gold nanoparticles (AuNPs), and then administered via tail vein injection to carotid atherosclerosis models for in vivo fluorescence imaging. Additionally, carotid tissues and cultured macrophages were analysed for nanoprobe binding, and MMP-14 and inflammation-related marker expression was evaluated by polymerase chain reaction, western blotting, and immunohistochemistry.Results: MMP-14 expression significantly increased with plaque progression, along with the upregulation of MMP-2 and inflammatory M1 markers, CD68 and F4/80, and significant downregulation of the M2 marker CD206. All of cell, tissue and in vivo fluorescence imaging exhibited a favourable targeting efficacy of [email protected] for MMP-14.Conclusions: MMP-14, a cell membrane-anchoring enzyme, can serve as a biomarker of vulnerable plaques, and MMP-14 substrate-based [email protected], with an intense fluorescence signal after activation and good biocompatibility, can be applied to screen for and monitor plaque progression in vivo.

scholarly journals Improved in Vivo Whole-Animal Detection Limits of Green Fluorescent Protein–Expressing Tumor Lines by Spectral Fluorescence Imaging

2007 ◽  
Vol 6 (4) ◽  
pp. 7290.2007.00023 ◽  
Author(s):  
Jenny M. Tam ◽  
Rabi Upadhyay ◽  
Mikael J. Pittet ◽  
Ralph Weissleder ◽  
Umar Mahmood
Keyword(s):  
Green Fluorescent Protein ◽  
Detection Limit ◽  
Cell Tracking ◽  
Fluorescent Protein ◽  
Spectral Imaging ◽  
Signal Interference ◽  
Fluorescence Signal ◽  
Emission Signal ◽  
Green Fluorescent

Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 × 103 cells for spectral imaging versus 1 × 106 cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.

scholarly journals Feasibility, sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo

2003 ◽  
Vol 7 (6) ◽  
pp. 765-773 ◽  
Author(s):  
Severine Wack ◽  
Amor Hajri ◽  
Francine Heisel ◽  
Malgorzata Sowinska ◽  
Cedric Berger ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Imaging ◽  
Fluorescent Protein ◽  
Laser Induced Fluorescence ◽  
Green Fluorescent

scholarly journals Thrombospondin-4, an extracellular matrix protein expressed in the developing and adult nervous system promotes neurite outgrowth.

1995 ◽  
Vol 131 (4) ◽  
pp. 1083-1094 ◽  
Author(s):  
S Arber ◽  
P Caroni
Keyword(s):  
Skeletal Muscle ◽  
Extracellular Matrix ◽  
Nervous System ◽  
Neuromuscular Junction ◽  
Neurite Outgrowth ◽  
Adult Skeletal Muscle ◽  
Wide Range ◽  
Thrombospondin 4

Extracellular matrix (ECM) molecules are involved in multiple aspects of cell-to-cell signaling during development and in the adult. In nervous system development, specific recognition processes, e.g., during axonal pathfinding and synaptogenesis involve modulation and signaling by ECM components. Much less is known about their presence and possible roles in the adult nervous system. We now report that thrombospondin-4 (TSP-4), a recently discovered member of the TSP gene family is expressed by neurons, promotes neurite outgrowth, and accumulates at the neuromuscular junction and at certain synapse-rich structures in the adult. To search for muscle genes that may be involved in neuromuscular signaling, we isolated cDNAs induced in adult skeletal muscle by denervation. One of these cDNAs coded for the rat homologue of TSP-4. In skeletal muscle, it was expressed by muscle interstitial cells. The transcript was further detected in heart and in the developing and adult nervous system, where it was expressed by a wide range of neurons. An antiserum to the unique carboxyl-terminal end of the protein allowed to specifically detect TSP-4 in transfected cells in vitro and on cryostat sections in situ. TSP-4 associated with ECM structures in vitro and in vivo. In the adult, it accumulated at the neuromuscular junction and at synapse-rich structures in the cerebellum and retina. To analyze possible activities of TSP-4 towards neurons, we carried out coculture experiments with stably transfected COS cells and motor, sensory, or retina neurons. These experiments revealed that TSP-4 was a preferred substrate for these neurons, and promoted neurite outgrowth. The results establish TSP-4 as a neuronal ECM protein associated with certain synapse-rich structures in the adult. Its activity towards embryonic neurons in vitro and its distribution in vivo suggest that it may be involved in local signaling in the developing and adult nervous system.

scholarly journals Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo

2015 ◽  
Vol 25 (2) ◽  
pp. 308-315 ◽  
Author(s):  
Dan Yu ◽  
Zhiqiang Dong ◽  
William Clay Gustafson ◽  
Rubén Ruiz-González ◽  
Luca Signor ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Rational Design ◽  
Fluorescent Protein ◽  
Red Fluorescent Protein

scholarly journals NGF-stimulated retrograde transport of trkA in the mammalian nervous system.

1995 ◽  
Vol 130 (1) ◽  
pp. 149-156 ◽  
Author(s):  
M D Ehlers ◽  
D R Kaplan ◽  
D L Price ◽  
V E Koliatsos
Keyword(s):  
Nervous System ◽  
Sciatic Nerve ◽  
Western Blot ◽  
Blot Analysis ◽  
Retrograde Transport ◽  
Model System ◽  
Specific Antibodies ◽  
Ngf Receptor ◽  
Ligation Site

The present study was designed to clarify the in vivo function of trkA as an NGF receptor in mammalian neurons. Using the rat sciatic nerve as a model system, we examined whether trkA is retrogradely transported and whether transport is influenced by physiological manipulations. Following nerve ligation, trkA protein accumulates distal to the ligation site as shown by Western blot analysis. The distally accumulating trkA species were tyrosine phosphorylated. The trkA retrograde transport and phosphorylation were enhanced by injecting an excess of NGF in the footpad and were abolished by blocking endogenous NGF with specific antibodies. These results provide evidence that, upon NGF binding, trkA is internalized and retrogradely transported in a phosphorylated state, possibly together with the neurotrophin. Furthermore, our results suggest that trkA is a primary retrograde NGF signal in mammalian neurons in vivo.

Imaging Molecular Targeting In Living Neural Cultures

1999 ◽  
Vol 5 (S2) ◽  
pp. 1228-1229
Author(s):  
Christopher S. Wallace ◽  
Michael A. Silverman ◽  
Michelle A. Burack ◽  
Janis E. Lochner ◽  
Richard G. Allen ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Hippocampal Neurons ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Complex Structure ◽  
Time Lapse ◽  
The Central Nervous System

Recent technical advances in the ability to attach an endogenously fluorescent protein sequence—i.e., green fluorescent protein or GFP and its derivatives--to any protein of experimental interest promises to mark a new era of progress in the study of protein targeting. Bringing these new tools to bear on neurons of the central nervous system has been challenging, however, because they have a very complex structure and are relatively difficult to transfect because they are post-mitotic.We use two cell culture approaches to characterize protein trafficking within neurons of the central nervous system in vitro. The first is a dissociated culture of hippocampal neurons from embryonic (El8) rats which is especially suited to analysis by conventional light microscopy because these neurons are grown on glass coverslips at low density. Neurons cultured in this way develop a morphology comparable to that seen in vivo and permit the establishment of axons and dendrites to be analyzed by time-lapse microscopy.

scholarly journals Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1195
Author(s):  
Przemysław Wieczorek ◽  
Marta Budziszewska ◽  
Patryk Frąckowiak ◽  
Aleksandra Obrępalska-Stęplowska
Keyword(s):  
Fluorescent Protein ◽  
Basic Research ◽  
Cellular Tissue ◽  
Fluorescence Signal ◽  
Reading Frame ◽  
Infectious Clones ◽  
Green Fluorescent ◽  
Virus Interactions ◽  
New Generation

Green fluorescent protein (GFP)-tagged viruses are basic research tools widely applied in studies concerning molecular determinants of disease during virus infection. Here, we described a new generation of genetically stable infectious clones of tomato torrado virus isolate Kra (ToTVpJL-Kra) that could infect Nicotiana benthamiana and Solanum lycopersicum. Importantly, a modified variant of the viral RNA2—with inserted sGFP (forming, together with virus RNA1, into ToTVpJL-KraGFP)—was engineered as well. RNA2 of ToTVpJL-KraGFP was modified by introducing an additional open reading frame (ORF) of sGFP flanked with an amino acid-coding sequence corresponding to the putative virus protease recognition site. Our further analysis revealed that sGFP-tagged ToTV-Kra was successfully passaged by mechanical inoculation and spread systemically in plants. Therefore, the clone might be applied in studying the in vivo cellular, tissue, and organ-level localization of ToTV during infection. By performing whole-plant imaging, followed by fluorescence and confocal microscopy, the presence of the ToTVpJL-KraGFP-derived fluorescence signal was confirmed in infected plants. All this information was verified by sGFP-specific immunoprecipitation and western blot analysis. The molecular biology of the torradovirus-plant interaction is still poorly characterized; therefore, the results obtained here opened up new possibilities for further research. The application of sGFP-tagged virus infectious clones and their development method can be used for analyzing plant-virus interactions in a wide context of plant pathology.

scholarly journals The role of retinoic acid in the formation and modulation of invertebrate central synapses

2017 ◽  
Vol 117 (2) ◽  
pp. 692-704 ◽  
Author(s):  
Cailin M. Rothwell ◽  
Eric de Hoog ◽  
Gaynor E. Spencer
Keyword(s):  
Nervous System ◽  
Retinoic Acid ◽  
Vitamin A ◽  
Neurite Outgrowth ◽  
Synapse Formation ◽  
Lymnaea Stagnalis ◽  
Trophic Factors ◽  
Electrical Synapses ◽  
Trophic Effect ◽  
Network Function

Trophic factors can influence many aspects of nervous system function, such as neurite outgrowth, synapse formation, and synapse modulation. The vitamin A metabolite, retinoic acid, can exert trophic effects to promote neuronal survival and outgrowth in many species and is also known to modulate vertebrate hippocampal synapses. However, its role in synaptogenesis has not been well studied, and whether it can modulate existing invertebrate synapses is also not known. In this study, we first examined a potential trophic effect of retinoic acid on the formation of excitatory synapses, independently of its role in neurite outgrowth, using cultured neurons of the mollusc Lymnaea stagnalis. We also investigated its role in modulating both chemical and electrical synapses between various Lymnaea neurons in cell culture. Although we found no evidence to suggest retinoic acid affected short-term synaptic plasticity in the form of post-tetanic potentiation, we did find a significant cell type-specific modulation of electrical synapses. Given the prevalence of electrical synapses in invertebrate nervous systems, these findings highlight the potential for retinoic acid to modulate network function in the central nervous system of at least some invertebrates. NEW & NOTEWORTHY This study performed the first electrophysiological analysis of the ability of the vitamin A metabolite, retinoic acid, to exert trophic influences during synaptogenesis independently of its effects in supporting neurite outgrowth. It was also the first study to examine the ability of retinoic acid to modify both chemical and electrical synapses in any invertebrate, nonchordate species. We provide evidence that all-trans retinoic acid can modify invertebrate electrical synapses of central neurons in a cell-specific manner.

scholarly journals Flavin Mononucleotide-Based Fluorescent Reporter Proteins Outperform Green Fluorescent Protein-Like Proteins as Quantitative In Vivo Real-Time Reporters

2010 ◽  
Vol 76 (17) ◽  
pp. 5990-5994 ◽  
Author(s):  
Thomas Drepper ◽  
Robert Huber ◽  
Achim Heck ◽  
Franco Circolone ◽  
Anne-Kathrin Hillmer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Flavin Mononucleotide ◽  
Fluorescence Signal ◽  
Reporter Protein ◽  
Reporter Proteins ◽  
Green Fluorescent

ABSTRACT Fluorescent proteins of the green fluorescent protein (GFP) family are commonly used as reporter proteins for quantitative analysis of complex biological processes in living microorganisms. Here we demonstrate that the fluorescence signal intensity of GFP-like proteins is affected under oxygen limitation and therefore does not reflect the amount of reporter protein in Escherichia coli batch cultures. Instead, flavin mononucleotide (FMN)-binding fluorescent proteins (FbFPs) are suitable for quantitative real-time in vivo assays under these conditions.

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