carbocyanines and Ischemia

The availability of transgenic disease backgrounds and the accessibility of molecular research reagents have contributed to make the mouse ischemic hindlimb the model of choice for many studies of angiogenesis, and to investigate new treatments for peripheral artery disease. A limitation of these models involves our inability to easily visualize the regenerated vascular architecture. Approaches such as micro-computed tomography and micro-angiography are expensive, technically demanding and not available to many laboratories. Here we describe a rapid and inexpensive adaptation of a vascular staining procedure for precise imaging of the mouse hindlimb vasculature. We introduced two technical modifications and an analytical extension to the original method including (i) pre-skinning of the muscle prior to fixation that preserves tissue integrity, (ii) mild pressure-desiccation subsequent to fixing that enhances resolution and image penetration, and (iii) reconstruction of confocal data into 3D images. The procedure provides resolution that is equivalent or superior to other approaches at a fraction of the cost, time and technology required.

Topics: Animals; Carbocyanines; Hindlimb; Imaging, Three-Dimensional; Ischemia; Mice; Mice, Inbred BALB C; Microscopy, Confocal

Expression of vascular endothelial growth factor receptors (VEGFRs) increases in ischemic muscles, and thus, VEGFR could potentially be used as marker to detect ischemia. Here, we investigated whether (99m)Tc or Cy5.5-labeled chitosan-DC101 conjugates could identify VEGFR-2 overexpressed in ischemia. To this end, chitosan was conjugated with the DC101 antibody and Cy5.5, FITC, or the HYNIC chelator for (99m)Tc-labeling. Targeting of the conjugate was evaluated in vitro and in vivo through cell-binding studies and gamma/optical imaging, respectively. A hindlimb ischemic mouse model was surgically created by femoral artery occlusion. The chitosan-DC101 conjugates exhibited VEGFR-selective cell binding properties as determined by both confocal microscopy and flow cytometry. At postoperative times of 2, 12, and 24 h, (99m)Tc or Cy5.5-labeled chitosan-DC101 conjugates were intravenously injected into the mice, and gamma/optical imaging studies were conducted at 1 or 3 h. Both the gamma and optical imaging results indicated a significantly higher uptake in ischemic muscles when compared with the contralateral nonischemic muscle. Further, semiquantitative analysis of scintigraphic imaging data revealed that the ischemic to contralateral limb ratio was 4.5 +/- 0.25 at 24 h postoperation. Western blotting analysis confirmed VEGFR expression in the ischemic muscle. In conclusion, we believe that (99m)Tc or Cy5.5-labeled chitosan-DC101 conjugates have the potential to be useful as VEGFR-2-targeted imaging agents for monitoring ischemia.

Topics: Animals; Antibodies, Monoclonal; Carbocyanines; Cells, Cultured; Chitosan; Female; Hindlimb; Humans; Ischemia; Mice; Mice, Inbred BALB C; Microvessels; Molecular Imaging; Muscle, Skeletal; Neovascularization, Physiologic; Protein Isoforms; Radionuclide Imaging; Receptors, Vascular Endothelial Growth Factor; Technetium

Ischemic tolerance is an endogenous neuroprotective mechanism in brain and other organs, whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia. Although many molecular mechanisms mediate delayed (gene-mediated) ischemic tolerance, the mechanisms underlying rapid (protein synthesis-independent) ischemic tolerance are relatively unknown. Here we describe a novel mechanism for the induction of rapid ischemic tolerance mediated by the ubiquitin-proteasome system. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors [carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), MG115 (carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal), and clasto-lactacystin-beta-lactone]. A proteomics strategy was used to identify ubiquitinated proteins after preconditioning ischemia. We focused our studies on two actin-binding proteins of the postsynaptic density that were ubiquitinated after rapid preconditioning: myristoylated, alanine-rich C-kinase substrate (MARCKS) and fascin. Immunoblots confirm the degradation of MARCKS and fascin after preconditioning ischemia. The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density and transient retraction of dendritic spines. This rapid and reversible synaptic remodeling reduced NMDA-mediated electrophysiological responses and renders the cells refractory to NMDA receptor-mediated toxicity. The dendritic spine retraction and NMDA neuroprotection after preconditioning ischemia are blocked by actin stabilization with jasplakinolide, as well as proteasome inhibition with MG132. Together these data suggest that rapid tolerance results from changes to the postsynaptic density mediated by the ubiquitin-proteasome system, rendering neurons resistant to excitotoxicity.

Topics: Analysis of Variance; Animals; Animals, Newborn; Carbocyanines; Carrier Proteins; Cell Death; Cells, Cultured; Cerebral Cortex; Enzyme Inhibitors; Glucose; Hypoxia; Intracellular Signaling Peptides and Proteins; Ischemia; Ischemic Preconditioning; Membrane Potentials; Membrane Proteins; Microfilament Proteins; Myristoylated Alanine-Rich C Kinase Substrate; Neurons; Patch-Clamp Techniques; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Synapses; Time Factors; Ubiquitin

Ischemia-reperfusion injury is a major complication occurring in acute myocardial infarction, cardiopulmonary bypass surgery, and heart transplantation. The aim of this study was to identify proteins that were involved in ischemia-reperfusion injury using fluorescence two-dimensional difference gel electrophoresis. We compared the 100,000 x g precipitate fractions of normal, ischemic and ischemia-reperfused rat hearts and detected six spots which changed more than two-fold in expression level and two additional spots related to these spots. Using peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, we identified five of these spots as protein disulfide isomerase A3 (PDA3), one as 60 kDa heat shock protein (HSP60) and two as elongation factor Tu (EF-Tu). HSP60 was increased during ischemia and decreased to normal expression level after reperfusion. EF-Tu was increased in ischemia but not decreased by reperfusion. We also found that several protein spots of PDA3 shifted towards a higher isoelectric point in ischemia and ischemia-reperfusion. Our data strongly suggested that PDA3 underwent dephosphorylation during ischemia and reperfusion and serine 343 of PDA3 was one of the phosphorylation sites.

Topics: Animals; Binding Sites; Carbocyanines; Chaperonin 60; Databases as Topic; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Image Processing, Computer-Assisted; Ischemia; Male; Microscopy, Fluorescence; Myocardium; Peptide Elongation Factor Tu; Phosphorylation; Protein Disulfide-Isomerases; Proteome; Rats; Rats, Wistar; Reperfusion Injury; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subcellular Fractions