Nerve growth factor reduces myocardial ischemia/reperfusion injury in rat hearts

Abstract

Background: Nerve growth factor (NGF) is a neurotrophin that supports the survival and differentiation of sympathetic neurons, and its increased expression after myocardial infarct was correlated with cardiac sympathetic hyperinnervation and arrhythmias. However, it is unclear whether NGF protects the heart during infarct. In this study, we sought to address this issue in rat heart exposed to ischemia/reperfusion injury (IRI).

Methods: NGF was administered intravenously (IV), 15 min before ischemia, at different concentrations in the absence or presence of inhibitors of phosphatidylinositol-3 kinase (PI3K) or nitric oxide synthase (NOS) in different groups of rats (n=6) with left coronary occlusion for 30 min followed by 120-min reperfusion. The area at risk and infarct to risk ratios were determined from sections stained with 1% 2,3,5-triphenylterazolium chloride.

Results: NGF treatment at doses of 0.015-15 μg/kg, with an optimal dose of 0.15 μg/kg given IV before ischemia, reduced the infarct size from about 60% at the area of risk to about 25%, indicating cardioprotection by about 60%. The infarct-sparing effects of NGF were partially abolished by the inhibition of PI3K and NOS using wortmannin and N(G)-monomethyl-l-arginine, respectively.

Conclusions: We have demonstrated for the first time that NGF attenuates myocardial infarct damage in an in vivo rat model of myocardial regional IRI. This cardioprotective effect is proposed to be related to the activities of PI3K and NOS. This suggests that NGF has a potential therapeutic role in the treatment of IRI.

Figure 1. NGF-induced cardioprotection (A, B) and hemodynamic values of mean arterial pressure (C) and heart rate (D) in a rat myocardial ischemia/reperfusion model

An in vivo anesthetized rat model was used for these experiments, with the general surgical protocol and determination of infarct size as described previously [7]. Briefly, thiobutabarbital sodium (inactin, 100 mg/kg, IP) was used for anesthesia, followed by a tracheotomy and artificial ventilation. Male Sprague-Dawley rats at 8 weeks of age (received humane care in compliance with the Guide for the Care and Use of Laboratory Animals; experiments were approved by the Ethical Committee of Medical College of Wisconsin) were ventilated with room air at 35–45 breaths/min supplemented with O₂. Arterial pH, pCO₂, and pO₂ were monitored and maintained throughout the experiment using an ABL 80 Flex pH/Blood Gas Analyzer. Body temperature was maintained at 37°C using a heating pad. The left common carotid artery was cannulated for blood pressure, heart rate, and blood gas measurements. A thoracotomy was performed at the fifth intercostal space, the pericardium was excised, and a ligature was placed around the left anterior descending coronary artery. Following surgical intervention and stabilization, rats were separated into groups and subjected to 30 min of ischemia and 120 min of reperfusion. The myocardium was made ischemic by placing the two ends of the ligature around the left anterior descending coronary artery through a polypropylene tube and fixing the tube to the epicardial surface with a hemostat. Removal of the hemostat allowed for reperfusion of the AAR. After 2 h of reperfusion, the ligature was again re-occluded and 0.5% Evans’ blue was perfused into the aortic cannula. The AAR was determined by the blue negative staining. The left ventricle was excised and cross-sectioned into four to five slices and further separated into the normal zone (LVA) and AAR. Slices were incubated in 1% 2,3,5-triphenylterazolium chloride to determine the infarct size. The heart was incubated overnight in 10% formaldehyde, and the infarcted tissue was dissected from the AAR and measured gravimetrically. Infarct size was expressed as a percentage of the AAR (part A) or the LVA (part B). Rats were treated IV with either saline or increasing doses of βNGF (Alomone Labs, Jerusalem, Israel) administered as an IV bolus, 15 min before 30-min ischemia followed by 120-min reperfusion. The inhibitors were injected 15 min before NGF treatment. Data are mean±SD of infarct size, n=6/group; *p<0.05 vs. control; **p<0.05 vs. 0.15 μg/kg NGF.