Name: intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels
Uploaded: 2013-01-04T12:00:00
Description: Watch this Scientific Journal Video about Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels at JoVE.com

We would like to thank Britta Kaltwasser for her excellent technical assistance and Mahesh Kumar Teli to organize the video filming preparation.

1. Animals
<ol>
	<li>Experiments were carried out according to the NIH guidelines for the care and use of laboratory animals and approved by local authorities. For all experiments, C57Bl6/J wild type mice, ApolipoproteinE-/- (ApoE KO) and SV129 mice (12-16 weeks old, 26-30 g body weight, 5-6 animals per experimental group) were used.</li>
</ol>
2. Staining of Cerebral Vessels with Colored Latex
<ol>
	<li>Prepare a mixture of 25 &mu;l carbon black ink (Herlitz, Germany) with 0.5 ml o...

<ol>
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Perfusion of CB1+CB2 by manual injection can be carried out successfully by without intensive training as it does not involve any specific device to imply certain pressure2,3. The heterogeneity of perfusion outcomes in our protocol is also negligible. Only 1 animal out of 16 non-ischemic animals and 3 out of 20 ischemic animals have showed incomplete perfusion. In these cases, incorporation of bubbles during saline perfusion leading to occlusion of vessels was most likely the reason of the unsuccessful outcome...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company Name</td>
 <td>Catalog No.</td>
 </tr>
 <tr>
 <td>Scribtol Schwarz (CB2)</td>
 <td>Pelican, Germany </td>
 <td>221 135</td>
 </tr>
 <tr>
 <td>Stempelfarbe (CB1)</td>
 <td>Herlitz PBS AG, Germany</td>
 <td>10417202</td>
 </tr>
 <tr>
 <td>Gedeo Latex </td>
 <td>Pebeo, France</td>
 <td>13042B</td>
 </tr>
 </tbody>
</table>

intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The cerebral vasculature dynamically responds to various pathophysiological states and it exhibits considerable differences between strains and under conditions of genetic manipulations. Essentially, a reliable technique for intracranial vessel staining is essential in order to study the pathogenesis of ischemic stroke. Until recently, a set of different techniques has been employed to visualize the cerebral vasculature including injection of low viscosity resin, araldite F, gelatin mixed with various dyes1 (i.e. carmine red, India ink) or latex with2 or without3 carbon black. Perfusion of white latex compound through the ascending aorta has been first reported by Coyle and Jokelainen3. Maeda et al.2 have modified the protocol by adding carbon black ink to the latex compound for improved contrast visualization of the vessels after saline perfusion of the brain. However, inefficient perfusion and inadequate filling of the vessels are frequently experienced due to high viscosity of the latex compound4. Therefore, we have described a simple and cost-effective technique using a mixture of two commercially available carbon black inks (CB1 and CB2) to visualize the cerebral vasculature in a reproducible manner5. We have shown that perfusion with CB1+CB2 in mice results in staining of significantly smaller cerebral vessels at a higher density in comparison to latex perfusion5. Here, we describe our protocol to identify the anastomotic points between the anterior (ACA) and middle cerebral arteries (MCA) to study vessel variations in mice with different genetic backgrounds. Finally, we demonstrate the feasibility of our technique in a transient focal cerebral ischemia model in mice by combining CB1+CB2-mediated vessel staining with TTC staining in various degrees of ischemic injuries.

The protocol described here overcomes the technical limitations of conventional latex based visualization of rodent cerebral vasculature. Figure 1A shows that following perfusion of the colored latex, only the large vessels on the ventral surface are stained, leaving the entire dorsal surface unstained. The outcome is also highly variable. Only one animal out of six shows partial staining of the ACA and the MCA visible on the dorsal surface of the brain (data not shown). Conversely, CB1+CB2 perfusion res...

Watch this Scientific Journal Video about Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels at JoVE.com

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Analysis of rodent cerebrovascular anatomy plays an important role in experimental stroke research. In this context, intravascular perfusion with colored latex has been considered as a standard tool for several years. However, this technique implies distinct technical limitations, which undermine its reproducibility. Here, we describe a simple method to visualize cerebral vessels in a reproducible manner. Injection of a mixture of two commercially available carbon black inks through the left myocardial ventricle results in adequate filling of cerebral vessels with high contrast visualization. We have successfully applied this technique to identify anastomotic points between cerebral vascular territories of mice with different genetic backgrounds. We finally give evidence that this novel and simple method for vessel staining can be combined with triphenyltetrazolium chloride (TTC) staining - a widely used tool to observe and analyze infarct volumes in mice.

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

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