Name: a method for 2-photon imaging of blood flow in the neocortex through a cranial window
Uploaded: 2008-02-25T14:50:00
Description: Watch this Scientific Journal Video about A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window at JoVE.com

<p class="jove_step">Tail vein injections of fluorescent dextrans dyes</p>
<ol>
<li>In order to image cerebral vasculature, animals must be injected intravenously with a fluorescent dye. We use dextran-conjugated dyes because the dextran moiety prevents the dye from crossing the brain blood barrier and leaking out of the blood vessels.</li>
<li>Mice are anesthetized with isoflurane (4% for induction, 1.5-2% during the injection).</li>
<li>Tail is disinfected with 70% alcohol.</li>
<li>With a 26 gauge needle, 75-100 &micro;l of a 5% v/v solution...

<ol>
	<li>Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex.">Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.</a> <em style='font-style: italic'>Proc Nat Acad Sci</em>. <b>95</b>, 15741-15746 (1998).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> <em style='font-style: italic'>Biomed. Eng. Online</em>. <b>17</b>, 2-13 (2003).</li><li>Zhang, S., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+impact+of+cortical+microcirculation+on+synaptic+structure+and+sensory-evoked+hemodynamic+responses+in+vivo.">Imaging the impact of cortical microcirculation on synaptic structure and sensory-evoked hemodynamic responses in vivo.</a> <b>5</b>, (2007).</li><li>Nishimura, N., Schaffer, C. B., Friedman, B., Lyden, P. D., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetrating+arterioles+are+a+bottleneck+in+the+perfusion+of+neocortex.">Penetrating arterioles are a bottleneck in the perfusion of neocortex.</a> <em style='font-style: italic'>Proc Nat Acad Sci</em>. <b>104</b>, 365-370 (2007).</li></ol>

<p class="jove_content">Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with two-photon microscopy. This video showed a method for how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.</p>

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<td>Fluorescein isothio-cyanate-dextran</td>
<td>Reagent</td>
<td>Sigma</td>
<td>FD500S</td>
<td>mol wt 500,000</td>
</tr>
<tr>
<td>Rhodamine B isothio-cyanate-dextran</td>
<td>Reagent</td>
<td>Simga</td>
<td>R9379</td>
<td>mol wt ~70,000
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a method for 2-photon imaging of blood flow in the neocortex through a cranial window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Watch this Scientific Journal Video about A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window at JoVE.com

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

<p class="jove_content">Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.</p>

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

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