Assessment of DNA replication in central nervous system by Laser Scanning Cytometry

2004 ◽  
Author(s):  
Dominik Lenz ◽  
Birgit Mosch ◽  
Jozsef Bocsi ◽  
Thomas Arendt ◽  
Attila Tßrnok
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Dna Replication ◽  
Laser Scanning ◽  
Laser Scanning Cytometry

scholarly journals Infection of Cells with Human Cytomegalovirus during S Phase Results in a Blockade to Immediate-Early Gene Expression That Can Be Overcome by Inhibition of the Proteasome

2002 ◽  
Vol 76 (11) ◽  
pp. 5369-5379 ◽  
Author(s):  
Elizabeth A. Fortunato ◽  
Veronica Sanchez ◽  
Judy Y. Yen ◽  
Deborah H. Spector
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Dna Content ◽  
Laser Scanning ◽  
Small Population ◽  
S Phase ◽  
Early Gene ◽  
Immediate Early ◽  
Laser Scanning Cytometry

ABSTRACT Cells infected with human cytomegalovirus (HCMV) after commencing DNA replication do not initiate viral immediate-early (IE) gene expression and divide before arresting. To determine the nature of this blockade, we examined cells that were infected 24 h after release from G0 using immunofluorescence, laser scanning cytometry, and fluorescence-activated cell sorting (FACS) analysis. Approximately 40 to 50% of the cells had 2N DNA content, became IE+ in the first 12 h, and arrested. Most but not all of the cells with >2N DNA content did not express IE antigens until after mitosis. To define the small population of IE+ cells that gradually accumulated within the S and G2/M compartments, cells were pulsed with bromodeoxyuridine (BrdU) just prior to S-phase infection and analyzed at 12 h postinfection for IE gene expression, BrdU positivity, and cell cycle position. Most of the BrdU+ cells were IE− and had progressed into G2/M or back to G1. The majority of the IE+ cells in S and G2/M were BrdU−. Only a few cells were IE+ BrdU+, and they resided in G2/M. Multipoint BrdU pulse-labeling revealed that, compared to cells actively synthesizing DNA at the beginning of the infection, a greater percentage of the cells that initiated DNA replication 4 h later could express IE antigens and proceed into S. Synchronization of the cells with aphidicolin also indicated that the blockade to the activation of IE gene expression was established in cells soon after initiation of DNA replication. It appears that a short-lived protein in S-phase cells may be required for IE gene expression, as it is partially restored by treatment with the proteasome inhibitor MG132.

Confocal Laser Microscopy of Physiologically Characterized Fluorescently Labeled Central Nervous System Neurons

1988 ◽  
Vol 46 ◽  
pp. 274-275
Author(s):  
J.N. Turner ◽  
J. Swann ◽  
K. Smith ◽  
M. Siemens ◽  
D. Szarowski ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Laser Scanning ◽  
Tissue Sample ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Brain Slices ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Efficient Detection

Confocal laser scanning microscopy (CLSM) is capable of three-dimensional imaging of fluorescently labeled single cells. Efficient detection via a photomultiplier and optical sectioning with high rejection of light from other specimen levels make it possible to image cells surrounded by either labeled or unlabeled tissue. It is no longer necessary to restrict high resolution light microscopy to cultured cells or those near the surface of a tissue sample. Cells can be observed üin situ” in a physiologically characterized environment. Central nervous system neurons can be electrophysiologically characterized and then injected with a fluorescent dye such as lucifer yellow. The CLSM can excite the dye and image the fluorescent emission in thick tissue preparations (hundreds of micrometers) making possible a new approach to the correlation of physiology and anatomy.Brain slices 350 μm thick were obtained from hippocampus and inferior colliculus of immature rats and incubated in oxygenated artificial cerebrospinal fluid. Cells were penetrated with micropipets, characterized electrophysiologically and ionophoretically injected with 5% lucifer yellow in LiAc.

scholarly journals Heparin Inhibits Leukocyte Rolling in Pial Vessels and Attenuates Inflammatory Changes in a Rat Model of Experimental Bacterial Meningitis

1997 ◽  
Vol 17 (11) ◽  
pp. 1221-1229 ◽  
Author(s):  
Joerg R. Weber ◽  
Klemens Angstwurm ◽  
Thomas Rosenkranz ◽  
Ute Lindauer ◽  
Dorette Freyer ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Bacterial Meningitis ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Leukocyte Rolling ◽  
Central Nervous System Inflammation ◽  
Inflammatory Processes

Heparin is a natural proteoglycan that was first described in 1916. In addition to its well characterized effect on blood coagulation, it is becoming clear that heparin also modulates inflammatory processes on several levels, including the interference with leukocyte–endothelium interaction. Anecdotal observations suggest a better clinical outcome of heparin-treated patients with bacterial meningitis. The authors demonstrate that heparin, a glycosaminoglycan, inhibits significantly in the early phase of experimental pneumococcal meningitis the increase of 1) regional cerebral blood flow (125 ± 18 versus 247 ± 42%), 2) intracranial pressure (4.5 ± 2.0 versus 12.1 ± 2.2 mm Hg), 3) brain edema (brain water content: 78.23 ± 0.33 versus 79.49 ± 0.46%), and 4) influx of leukocytes (571 ± 397 versus 2400 ± 875 cells/μL) to the cerebrospinal fluid compared with untreated rats. To elucidate the possible mechanism of this observation, the authors investigated for the first time leukocyte rolling in an inflammatory model in brain venules by confocal laser scanning microscopy in vivo. Heparin significantly attenuates leukocyte rolling at 2, 3, and 4 hours (2.8 ± 1.3 versus 7.9 ± 3.2/100 μm/min), as well as leukocyte sticking at 4 hours (2.1 ± 0.4 versus 3.5 ± 1.0/100 μm/min) after meningitis induction compared with untreated animals. The authors conclude that heparin can modulate acute central nervous system inflammation and, in particular, leukocyte–endothelium interaction, a key process in the cascade of injury in bacterial meningitis. They propose to evaluate further the potential of heparin in central nervous system inflammation in basic and clinical studies.

scholarly journals Extravascular CX3CR1+Cells Extend Intravascular Dendritic Processes into Intact Central Nervous System Vessel Lumen

2013 ◽  
Vol 19 (4) ◽  
pp. 778-790 ◽  
Author(s):  
Deborah S. Barkauskas ◽  
Teresa A. Evans ◽  
Jay Myers ◽  
Agne Petrosiute ◽  
Jerry Silver ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Central Nervous System ◽  
Nervous System ◽  
Laser Scanning ◽  
Immune Cell ◽  
Inflammatory Responses ◽  
Tissue Injury ◽  
Critical Role ◽  
Laser Scanning Microscopy ◽  
Direct Access

AbstractWithin the central nervous system (CNS), antigen-presenting cells (APCs) play a critical role in orchestrating inflammatory responses where they present CNS-derived antigens to immune cells that are recruited from the circulation to the cerebrospinal fluid, parenchyma, and perivascular space. Available data indicate that APCs do so indirectly from outside of CNS vessels without direct access to luminal contents. Here, we applied high-resolution, dynamic intravital two-photon laser scanning microscopy to directly visualize extravascular CX3CR1+APC behavior deep within undisrupted CNS tissues in two distinct anatomical sites under three different inflammatory stimuli. Surprisingly, we observed that CNS-resident APCs dynamically extend their cellular processes across an intact vessel wall into the vascular lumen with preservation of vessel integrity. While only a small number of APCs displayed intravascular extensions in intact, noninflamed vessels in the brain and the spinal cord, the frequency of projections increased over days in an experimental autoimmune encephalomyelitis model, whereas the number of projections remained stable compared to baseline days after tissue injury such as CNS tumor infiltration and aseptic spinal cord trauma. Our observation of this unique behavior by parenchyma CX3CR1+cells in the CNS argues for further exploration into their functional role in antigen sampling and immune cell recruitment.

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