scholarly journals Identification and Characterization of Novel Perivascular Adventitial Cells in the Whole Mount Mesenteric Branch Artery Using Immunofluorescent Staining and Scanning Confocal Microscopy Imaging

2012 ◽  
Vol 2012 ◽  
pp. 1-6
Author(s):  
Chandra Somasundaram ◽  
Rahul K. Nath ◽  
Richard D. Bukoski ◽  
Debra I. Diz
Keyword(s):  
Confocal Microscopy ◽  
Neurite Growth ◽  
Immunofluorescent Staining ◽  
Neural Cells ◽  
Microscopy Imaging ◽  
Stress Injury ◽  
Scanning Confocal Microscopy ◽  
Adult Rat ◽  
Calcium Sensing ◽  
Whole Mount

A novel perivascular adventitial cell termed, adventitial neuronal somata (ANNIES) expressing the neural cell adhesion molecule (NCAM) and the vasodilator neuropeptide, calcitonin gene-related peptide (CGRP), exists in the adult rat mesenteric branch artery (MBA) in situ. In addition, we have previously shown that ANNIES coexpress CGRP and NCAM. We now show that ANNIES express the neurite growth marker, growth associated protein-43(Gap-43), palladin, and the calcium sensing receptor (CaSR), that senses changes in extracellular Ca(2+) and participates in vasodilator mechanisms. Thus, a previously characterized vasodilator, calcium sensing autocrine/paracrine system, exists in the perivascular adventitia associated with neural-vascular interface. Images of the whole mount MBA segments were analyzed under scanning confocal microscopy. Confocal analysis showed that the Gap-43, CaSR, and palladin were present in ANNIES about 37 ± 4%, 94 ± 6%, and 80 ± 10% respectively, comparable to CGRP (100%). Immunoblots from MBA confirmed the presence of Gap-43 (48 kD), NCAM (120 and 140 kD), and palladin (90–92 and 140 kD). In summary, CGRP, and NCAM-containing neural cells in the perivascular adventitia also express palladin and CaSR, and coexpress Gap-43 which may participate in response to stress/injury and vasodilator mechanisms as part of a perivascular sensory neural network.

scholarly journals Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC-ε to RACK2 in adult rat and mouse

2009 ◽  
Vol 297 (2) ◽  
pp. H718-H725 ◽  
Author(s):  
Richard A. Fenton ◽  
Satoshi Komatsu ◽  
Mitsuo Ikebe ◽  
Lynne G. Shea ◽  
James G. Dobson
Keyword(s):  
Confocal Microscopy ◽  
Phospholipase C ◽  
Adrenergic Agonist ◽  
Rat Cardiomyocytes ◽  
Scanning Confocal Microscopy ◽  
Adult Rat ◽  
Sds Page ◽  
T Tubules ◽  
Isolated Rat ◽  
Stimulation Of

Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A1 receptors (A1R) on the adrenergic signaling pathway. An indirect A1R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-ε to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A1R activation of PKC-ε induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A1R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-ε and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-ε to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A1R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-ε co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-ε with RACK2 by 61%. With rat cardiomyocytes, the β-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A1R stimulation of the heart is associated with PLC-initiated PKC-ε translocation and association with RACK2.

scholarly journals Live whole-eye, ex vivo imaging and laser-induced micro injury of the corneal basal epithelium and visualization of resident macrophage responses

2021 ◽  
Author(s):  
Sebastian M.D. Gulka ◽  
Ana M. Litke ◽  
Kerry R. Delaney ◽  
Robert L. Chow
Keyword(s):  
Epithelial Cell ◽  
Confocal Microscopy ◽  
Laser Power ◽  
Laser Scanning ◽  
Cell Damage ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy ◽  
High Laser ◽  
Whole Mount ◽  
Laser Induced Damage

Purpose: In this paper, we describe a novel live-imaging approach to visualize the short-term response of the mouse cornea to basal epithelial cell damage. Laser scanning confocal microscopy was used to induce precisely-defined, localized corneal basal epithelial cell damage and the live macrophage response to this damage was visualized and analyzed. Methods: Lipophilic fluorescent dyes, SGC5 or FM 4-64, were injected into the anterior chamber of enucleated eyes and imaged live in whole-mount using confocal laser scanning microscopy. Laser-induced damage was performed by focusing onto a defined region of the corneal basal epithelium for a brief period using a high laser power setting and then returning to low laser power for imaging. Eyes from CX3CR1+/GFP mice were used to observe macrophage responses to laser damage in real-time. Results: SGC5 or FM 4-64 dyes injected into the anterior chamber readily enter the cornea and are taken up by the stromal layer and labeled the outer membranes of corneal epithelial cells and remained stable when visualized using low laser power. Subjecting a defined region of the basal epithelium to high laser power for 1 minute or longer led to a rapid internalization of dye in the exposed basal epithelium cells and overall increase in cellular fluorescence. This change in fluorescence was also accompanied by cell swelling and contraction. Cellular internalization of the non-lipophilic, dye Alexa 647 hydrazide, indicated that membranes were compromised indicating that exposure to high power laser stimulation causes cellular damage to the basal epithelium. Visualization of corneal resident macrophages close to the site of laser-induced damage showed that within minutes, projecting macrophage filopodia extended towards the damaged region at a rate of 0.75um/min for roughly 40 minutes. Conclusion: We have developed a novel approach to image the live cornea and its response to damage. Laser-scanning confocal microscopy can be utilized to induce localized damage to mouse corneal basal epithelium and elicit a macrophage morphological response. This approach represents a useful tool for studying corneal wound healing and cellular responses to damage using live whole-mount imaging.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

Observation of Fine Structure in Bicontinuous Phase-Separated Domains of a Polymer Blend by Laser Scanning Confocal Microscopy

2001 ◽  
Vol 34 (15) ◽  
pp. 5186-5191 ◽  
Author(s):  
Hiroshi Jinnai ◽  
Hiroshi Yoshida ◽  
Kohtaro Kimishima ◽  
Yoshinori Funaki ◽  
Yoshitsugu Hirokawa ◽  
...  
Keyword(s):  
Fine Structure ◽  
Confocal Microscopy ◽  
Polymer Blend ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy

scholarly journals The Contraction of Smooth Muscle Cells of Intrapulmonary Arterioles Is Determined by the Frequency of Ca2+ Oscillations Induced by 5-HT and KCl

2005 ◽  
Vol 125 (6) ◽  
pp. 555-567 ◽  
Author(s):  
Jose F. Perez ◽  
Michael J. Sanderson
Keyword(s):  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Confocal Microscopy ◽  
Smooth Muscle Cells ◽  
Phase Contrast ◽  
Muscle Cells ◽  
Low Frequencies ◽  
Scanning Confocal Microscopy ◽  
Agonist Concentration ◽  
Mouse Lungs

Increased resistance of the small blood vessels within the lungs is associated with pulmonary hypertension and results from a decrease in size induced by the contraction of their smooth muscle cells (SMCs). To study the mechanisms that regulate the contraction of intrapulmonary arteriole SMCs, the contractile and Ca2+ responses of the arteriole SMCs to 5-hydroxytrypamine (5-HT) and KCl were observed with phase-contrast and scanning confocal microscopy in thin lung slices cut from mouse lungs stiffened with agarose and gelatin. 5-HT induced a concentration-dependent contraction of the arterioles. Increasing concentrations of extracellular KCl induced transient contractions in the SMCs and a reduction in the arteriole luminal size. 5-HT induced oscillations in [Ca2+]i within the SMCs, and the frequency of these Ca2+ oscillations was dependent on the agonist concentration and correlated with the extent of sustained arteriole contraction. By contrast, KCl induced Ca2+ oscillations that occurred with low frequencies and were preceded by small, localized transient Ca2+ events. The 5-HT–induced Ca2+ oscillations and contractions occurred in the absence of extracellular Ca2+ and were resistant to Ni2+ and nifedipine but were abolished by caffeine. KCl-induced Ca2+ oscillations and contractions were abolished by the absence of extracellular Ca2+ and the presence of Ni2+, nifedipine, and caffeine. Arteriole contraction was induced or abolished by a 5-HT2–specific agonist or antagonist, respectively. These results indicate that 5-HT, acting via 5-HT2 receptors, induces arteriole contraction by initiating Ca2+ oscillations and that KCl induces contraction via Ca2+ transients resulting from the overfilling of internal Ca2+ stores. We hypothesize that the magnitude of the sustained intrapulmonary SMC contraction is determined by the frequency of Ca2+ oscillations and also by the relaxation rate of the SMC.

Real-time confocal microscopy imaging of corneal cytoarchitectural changes induced by different stresses

2021 ◽  
pp. 108706
Author(s):  
Guoliang Wang ◽  
Xiaoya An ◽  
Xiaoping Zhou ◽  
Mengyi Jin ◽  
Xuemei Wang ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Real Time ◽  
Microscopy Imaging ◽  
Confocal Microscopy Imaging

scholarly journals The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy.

1994 ◽  
Vol 42 (11) ◽  
pp. 1413-1416 ◽  
Author(s):  
S L Erlandsen ◽  
E M Rasch
Keyword(s):  
Confocal Microscopy ◽  
Genome Size ◽  
Dna Content ◽  
Giardia Lamblia ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Evolutionary Development ◽  
Scanning Confocal Microscopy ◽  
Dividing Cells ◽  
C Value

We investigated direct measurement of the DNA content of the parasitic intestinal flagellate Giardia lamblia through quantitation by Feulgen microspectrophotometry and also by visualization of Feulgen-stained DNA chromosomes within dividing cells by laser scanning confocal microscopy. Individual trophozoites of Giardia (binucleate) contained 0.144 +/- 0.018 pg of DNA/cell or 0.072 pg DNA/nucleus. Giardia lamblia cysts (quadranucleate) contained 0.313 +/- 0.003 pg DNA or 0.078 pg DNA/nucleus. The genome size (C) value per nucleus ranged between 6.5-7.1 x 10(7) BP for trophozoites and cysts, respectively. Confocal microscopic examination of Giardia trophozoites undergoing binary fission revealed five chromosome-like bodies within each nucleus. Further information about genome size and DNA content within different Giardia species may help to clarify the pivotal role of these primitive eukaryotic cells in evolutionary development.

The Effect of Chloroquine on the Apoptosis Induced by Cisplatin in Human Gastric Cancer BGC823 Cells

2014 ◽  
Vol 926-930 ◽  
pp. 1124-1127
Author(s):  
Zhen Xun Jin ◽  
Li Li Zhang ◽  
Yan Wang ◽  
Lin Chuan Zeng ◽  
Yang Yu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Confocal Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Combination Group ◽  
Human Gastric Cancer ◽  
Apoptotic Cells ◽  
Toxic Dose ◽  
Scanning Confocal Microscopy ◽  
Mtt Tests

The aim of this study is to investigate the effects and mechanism of chloroquine (CQ) on the apoptosis induced by cisplatin in human gastric cancer BGC823 cells. MTT assay was used to detect the state of cell growth. The appearances of cellular apoptosis were detected by laser scanning confocal microscopy and light microscopy. The expressions of LC3 and p62 were detected by laser scanning confocal microscopy. MTT tests showed that the non-toxic dose of CQ could increase the inhibition rate of BGC823 cells induced by cisplatin. Under the light microscope, the ratio of apoptotic cells in the group treated with non-toxic dose of CQ combined with cisplatin was higher than that in the group treated with cisplatin alone. Hoechst33342 staining showed that the ratio of apoptotic cells in the combination group was higher than that in the cisplatin group. The expression and colocalization of LC3 and p62 proteins were significantly increased in the combination group. These results indicate that CQ can enhance the cell apoptosis induced by cisplatin in BGC823 cells, which is through the inhibition of autophagy.

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