Surgical Approach

Cardiac access

Both mice and rats can be anesthetized for open cardiac interventions with a cocktail of ketamine (mice, 100 mg/kg; rats, 95 mg/kg) and xylazine (mice, 10 mg/kg; rats, 4 mg/kg). This method of anesthesia requires artificial ventilation with room air (mice, 120-140 breaths/ min with a weight adjusted tidal volume of 0.2ml/breath; rats, 90-100 breaths/min with a weight adjusted tidal volume of 1.5-2.0 ml/breath) after endotracheal intubation. Upon performing a left thoracotamy (~4 cm), the heart can be accessed through a 1 cm incision of the intercostal muscle between the fourth and fifth ribs using rib retractors (mice, 3 cm; rats, 5 cm). The LAD artery is identified as the coronary system branching from the left circumflex just below the left atrial appendage.

Cardiac injection Cardiac angiogenesis can be modulated in different vascular beds of the cardiac myocardium, but the direct access approach facilitates interventions targeted at the anterior wall of the heart. To this end, a strategy based on the positive identification of the LAD is employed to direct the two 25 ^l injections (30 G x 8 mm syringe) of treatment (e.g., growth factor, cells, etc.) or vehicle alone is administered 2mm apart on the mid-left anterior ventricular wall (see Figure 78.2). After treatment the lungs are inflated, and the chest wall is closed with a three-layer closure. Sets of animals can then be sacrificed at different time points to histologically quantify cardiac angiogenesis, or be potentially imaged with noninvasive approaches currently being developed as measures of cardiac perfusion.

Assessment approach In the aging heart, both morphometric and immuno-histology protocols have been employed in the quantification of angiogenesis. Morphometric studies of perfusion fixed tissue can provide accurate assessment about cardiac capillary density as well as the macrovasculature in the heart (Anversa et al., 1991; Anversa et al., 1994). Immunohistology can differentiate the heterogeneous endothelial cell subpopulations that contribute to cardiac angiogenesis (Edelberg et al., 2002b; Cai et al., 2003;

Xaymardan et al., 2004b). Studies directed at the mid-papillary level in cross sections of the left ventriculature provide a direct assessment of the potential actions of local injections directed at the level of the mid LAD.

Morphometric studies Morphometric analysis employs hearts that are arrested in diastole with cadmium-Cl and cannulation-based perfusion formalin fixation (Li et al., 1997) and allows for the direct assessment of capillary profiles (Anversa et al., 1994). In addition to providing a quantification of capillary density, this approach facilitates the direct analysis of myocyte-capillary ratios and quantification of the left coronary arterial tree.

Immunohistology—Vascular histology The characterization of cardiac angiogenesis by immu-nohistology can be performed on perfusion fixed or embedded tissue. Immunostains with antibodies directed against platelet endothelial cell adhesion molecule (PECAM; CD31), as well as VE cadherin, can provide an overall assessment of vascular density in the hearts. In addition, studies of von Willebrand factor, VEGF receptors, including Flk-1 and Flt-1, Ang receptors, Tie-2 and PDGF receptors a and p can provide important insight into the kinetics and potential mechanisms governing alterations in angiogenic pathways in the aging heart. The analysis of capillary density can be determined by automated image analysis such as CapiShape (Hu et al., 1999) or directly through manual quantification of all stained luminal structures in highpower fields (40-100 x magnification) in a blinded approach.

Figure 78.2 This diagram of the heart illustrates the area at risk in the left ventricle resulting from the occlusion of the left anterior descending artery (LAD) in the rat myocardial infarction model. Note the site of injection and level of ligation.

In vivo anatomical/functional imaging Recent advances in noninvasive approaches employing vascular contrast with both echocardiography (ultrasound-based) and magnetic resonance imaging (NMR-based) offer the potential to provide important information about cardiac microvascular anatomy and blood flow after angiogenic induction (Pearlman et al., 1995; Wilke et al., 2000) as well as after myocardial infarction (Gladish, 2005; Kaandorp et al., 2005).

Myocardial Infarction Model

The significance of cardiac angiogenesis can be functionally measured in the cardioprotection provided in myocardial infarction models. Indeed, the ability to induce myocardial injury (ischemia, infarction, etc.) provides an important model of clinical cardiovascular disease and is a useful technique in studying cardiac-specific angiogenesis. Specifically, acute coronary occlusion models have been used to observe angiogenesis after the administration of growth factors (Edelberg et al., 2002b; Chachques et al., 2004; Xaymardan et al., 2004b), stem cells (Nagaya et al., 2004; Xaymardan et al., 2004a), and genes that encode for growth factors (Schwarz et al., 2000; Su et al., 2002; Hao et al., 2004; Hao et al., 2004; Su et al., 2004).

Surgical Approach

A left ventricular myocardial infarction can be induced via the occlusion of the LAD artery by physical ligation using an 8-0 nylon suture (Ethilon, Ethicon, NJ) or by photothrombosis (see Figure 78.2), which involves a systemic injection of a photosensitizing dye (e.g., Rose Bengal) and excitation of the interested artery with a laser ion light source as Rosen (2001) and Eichenbaum (2002) have displayed in mice. This procedure can be used in concert with the molecular injection procedure where administration of the treatments can be given prior to or after the occlusion. Moreover, the duration of the occlusion can be varied from periods of ischemia and reperfusion to permanent coronary ligation, modeling acute coronary syndrome and acute myocardial infarction, respectively. In addition, the functional significance of various interventions can be assessed at a range of time points after myocardial infarction to investigate the potential role of angiogenic pathways in the aging rodent heart.

Blood Pressure Health

Blood Pressure Health

Your heart pumps blood throughout your body using a network of tubing called arteries and capillaries which return the blood back to your heart via your veins. Blood pressure is the force of the blood pushing against the walls of your arteries as your heart beats.Learn more...

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