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

Mohammad R. Hasan; Josephine Herz; Dirk M. Hermann; Thorsten R. Doeppner

doi:10.3791/4374

Summary

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.

Abstract

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.

Protocol

1. Animals

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.

2. Staining of Cerebral Vessels with Colored Latex

Prepare a mixture of 25 μl carbon black ink (Herlitz, Germany) with 0.5 ml of latex compound (Pebeo, France) at a 1:20 ratio in an EP tube and warm up the mixture at 37 °C in a water bath. Collect the mixture in a 2 ml syr....