Focal Ischemic Stroke Models

Focal ischemic stroke models, whether in larger mammals such as cats, dogs, or nonhuman primates, or in small mammals such as rodents, usually involve occlusion of one MCA (Lipton 1999). Focal ischemia is differentiated from global ischemia in two ways. First, even at the core of the lesion, the blood flow is almost always higher than during global ischemia so that longer insults are required to cause damage. Second, there is a significant gradation of ischemia from the core of the lesion to its outermost boundary, and hence there are different metabolic conditions within the affected site. Because of its duration and heterogeneity, the insult is much more complex than global ischemia, but it is an invaluable model for stroke and is thus widely studied. There are two models of focal ischemic stroke: transient focal ischemia and permanent focal ischemia. In transient focal ischemia models, vessels are blocked for up to 3 h, followed by prolonged reperfusion, whereas in permanent focal ischemia, the arterial blockage is maintained throughout an experiment, usually for one or more days.

Transient middle cerebral artery occlusion There are two principal occlusion sites in this model (see Figure 79.1). In proximal occlusion, the MCA is occluded close to its branching from the internal carotid artery, before the origin of the lenticulostriate arteries. A newer and now widely used approach to proximal MCA occlusion is the insertion of a nylon suture into the carotid artery, past the point at which the MCA branches so that the latter is occluded at its origin.

The procedure is as follows. After a midline neck incision, the left external carotid and pterigoparatine arteries are isolated and ligated with silk thread. The internal carotid artery is occluded at the peripheral site of the bifurcation of the internal carotid artery (ICA) and the pterigoparatine artery with a small clip, and the common carotid artery (CCA) is ligated with silk thread. The external carotid artery (ECA) is cut, and a nylon monofilament, whose tip is blunted (0.20-0.22 mm for a mouse) with a coagulator, is inserted into the ECA. The ECA and the inserted nylon thread are tightened with a silk suture, which prevents bleeding during advancement of the nylon thread and during its removal at the time of reperfusion, and rotated for its advancement into the ICA. After removal of the clip at the ICA, the nylon thread is advanced until light resistance is felt and the distance from the nylon thread tip to the internal carotid artery-pterygophalatine artery bifurcation is slightly more than 6 mm (mouse), and the distance to the ICA-ECA bifurcation is slightly less than 9 mm. During MCAO, the parietal bone becomes pale on the occluded side and laser Doppler flowmetry reveals that blood flow in this area falls to less than 20% of baseline. The nylon thread and the CCA ligature are removed after an occlusion period and reperfusion occurs with release of blood flow from the internal carotid artery. There are some potentially important artifacts with this widely used method. Blood flow following temporary ischemia is somewhat compromised by the partial occlusion of the carotid arteries with the filament and there is quite extensive damage to small arteries in the ischemic field, with damage to endothelial and smooth muscle cells. It has been suggested that this damage may affect subsequent neuronal cell death by exacerbating the leukocyte response in the reperfusion period (Ishikawa et al., 2004).

Permanent middle cerebral artery occlusion The commonly used permanent focal ischemia model involves occlusion of one or more branches of the MCA. The MCA is exposed via a transtemporal approach. After the temporalis muscle is retracted, a 2- to 3-mm burr hole is drilled 2 to 3 mm rostral to the fusion of the zygoma and squamosal bones. The MCA is exposed after opening and retracting the dura matter. Using a steel hook maneuvered via a micromanipulator, the MCA is elevated and electrocoagulated (McAuley, 1995).

Embolic model of focal ischemia The procedure for this model is similar to the transient focal ischemic model (Zhang et al., 2003). Briefly, a longitudinal incision is made in the midline of the ventral cervical skin. The CCA, ICA, and ECA are exposed. The distal portion of the ECA is ligated with two sutures and the ECA is cut between these two sutures. A silk suture is tied loosely around the origin of the ECA. The CCA and ICA are clamped temporarily using micro-vascular clips. A small puncture is made on the wall of the

ECA with a pair of spring scissors. A modified PE-10 catheter connected with a PE-50 tubing (40 mm in length for 10 ml thrombus and 20 mm for 5 ml) filled with bovine thrombin is introduced into the lumen of the ECA via the puncture. The suture around the origin of the ECA is tightened and the clip on the ICA is removed. After the blood is withdrawn, the catheter is advanced up in the ICA until its tip is 1 to 2 mm away from the origin of the MCA. The catheter is retained there for 15 minutes to allow formation of a clot. Once the clot has formed, it is then gently injected into the MCA. The catheter in the ICA is removed five minutes after the clot injection and the ECA is ligated.

Photothrombotic distal MCA occlusion The use of a photochemical reaction to produce focal cortical ischemia in the rat brain was first described in 1985 (Watson et al., 1985). In this model, vascular thrombosis is induced by transcranial illumination with a filtered light source in combination with intravenous injection of a photosensitive dye. Electron microscopy and light microscopy studies showed intravascular thrombotic material, red blood cell stasis, and platelet aggregates adhering to luminal surfaces inside vessels and intravascular thrombosis that is responsible for the occurrence of ischemia leading to infarction. Increased permeability through disruption of the BBB by the photochemical reaction is also involved in this type of model. The method involves mounting the rat in a stereotaxic head-holder, and making a 1 to 1.5-cm vertical incision between the right eye and ear. With the aid of an operating microscope, a burr hole is made with a highspeed drill. Care should be taken not to injure the dura matter. The distal segment of the right MCA is thus exposed. A krypton laser operating at 568 nm (Innova 301, Coherent Inc, or 643-Y-A01, Melles Griot Inc) is used to irradiate the distal MCA at a power of 20 mW for four minutes. The laser beam is focused with a 30-cm focal length convex lens and positioned with a mirror onto the distal MCA. The photosensitizing dye, normally rose bengal or erythrosin B (15-25 mg/mL in 0.9% saline), is administered intravenously at a body dose of 20 mg/kg over 90 seconds, starting simultaneously with four minutes of laser irradiation.

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