William B. Wead

ATTENUATION OF HEART RATE CONTROL AND NEURAL DEGENERATION IN NUCLEUS AMBIGUUS FOLLOWING CHRONIC INTERMITTENT HYPOXIA IN YOUNG ADULT FISCHER 344 RATS

Chronic intermittent hypoxia (CIH) attenuates baroreflex control of heart rate (HR). In this study, we assessed whether CIH exposure reduced nucleus ambiguus (NA) control of HR and induced neural degeneration in the NA. Fischer 344 (age: 3-4 months) rats were exposed to either room air (RA: normoxia) or intermittent hypoxia for 35-50 days. At the end of these exposures, animals were anesthetized with pentobarbital. HR responses to arterial blood pressure (AP) changes induced by phenylephrine (PE) and sodium nitroprusside (SNP) were measured. In another set of rats, HR and AP responses to L-glutamate (L-Glu) microinjections (10 mM, 20 nl) into the left NA and electrical stimulation of the left cervical vagus nerve at 1-30 Hz (0.5 mA, 1 ms) for 20 s were measured. Brainstem slices at the level of -800, -400, 0, +400, +800 microm relative to the obex were processed in additional rats using Nissl staining. The NA was identified by retrogradely labeling vagal motoneurons using the tracer tetramethylrhodamine dextran (TMR-D) which was injected into the ipsilateral nodose ganglion. We found that CIH significantly 1) reduced the baroreflex control of HR (slope RA: -1.2+/-0.2 bpm/mmHg; CIH -0.5+/-0.1 bpm/mmHg; P<0.05); 2) attenuated the HR responses to l-Glu injections into the NA [HR: -280+/-15 (RA) vs. -235+/-16 (CIH) beats/min; P<0.05]; 3) augmented the HR responses to electrical stimulation of the vagus (P<0.05); 4) induced a significant cellular loss in the NA region (P<0.05). Thus, CIH induces a cell loss in the NA region which may contribute to attenuation of baroreflex sensitivity and NA control of HR following CIH.

Structural remodeling of nucleus ambiguus projections to cardiac ganglia following chronic intermittent hypoxia in C57BL/6J mice.

The baroreflex control of heart rate (HR) is reduced following chronic intermittent hypoxia (CIH). Since the nucleus ambiguus (NA) plays a key role in baroreflex control of HR, we examined whether CIH remodels vagal efferent projections to cardiac ganglia. C57BL/6J mice (3–4 months of age) were exposed to either room air (RA) or CIH for 3 months. Confocal microscopy was used to examine NA axons and terminals in cardiac ganglia following Fluoro‐Gold (FG) injections to label cardiac ganglia, and microinjections of tracer DiI into the left NA to anterogradely label vagal efferents. We found that: 1) Cardiac ganglia were widely distributed on the dorsal surface of the atria. Although the total number of cardiac ganglia did not differ between RA and CIH mice, the size of ganglia and the somatic area of cardiac principal neurons (PNs) were significantly decreased (P < 0.01), and the size of the PN nuclei was increased following CIH (P < 0.01). 2) NA axons entered cardiac ganglia and innervated PNs with dense basket endings in both RA and CIH mice, and the percentage of innervated PNs was similar (RA: 50 ± 1.0%; CIH: 49 ± 1.0%; P > 0.10). In CIH mice, however, swollen cardiac axons and terminals without close contacts to PNs were found. Furthermore, varicose endings around PNs appeared swollen and the axonal varicose area around PNs was almost doubled in size (CIH: 163.1 ± 6.4 μm2; RA: 88 ± 3.9 μm2, P < 0.01). Thus, CIH significantly altered the structure of cardiac ganglia and resulted in reorganized vagal efferent projections to cardiac ganglia. Such remodeling of cardiac ganglia and vagal efferent projections provides new insight into the effects of CIH on the brain–heart circuitry of C57BL/6J mice. J. Comp. Neurol. 509:103–117, 2008.

Chronic intermittent hypoxia impairs heart rate responses to AMPA and NMDA and induces loss of glutamate receptor neurons in nucleus ambiguus of F344 rats

Chronic intermittent hypoxia (CIH), as occurs in sleep apnea, impairs baroreflex-mediated reductions in heart rate (HR) and enhances HR responses to electrical stimulation of vagal efferent. We tested the hypotheses that HR responses to activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the nucleus ambiguous (NA) are reduced in CIH-exposed rats and that this impairment is associated with degeneration of glutamate receptor (GluR)-immunoreactive NA neurons. Fischer 344 rats (3-4 mo) were exposed to room air (RA) or CIH for 35-50 days (n = 18/group). At the end of the exposures, AMPA (4 pmol, 20 nl) and NMDA (80 pmol, 20 nl) were microinjected into the same location of the left NA (-200 microm to +200 microm relative to caudal end of area postrema; n = 6/group), and HR and arterial blood pressure responses were measured. In addition, brain stem sections at the level of -800, -400, 0, +400, and +800 microm relative to obex were processed for AMPA and NMDA receptor immunohistochemistry. The number of NA neurons expressing AMPA receptors and NMDA receptors (NMDARs) was quantified. Compared with RA, we found that after CIH 1) HR responses to microinjection of AMPA into the left NA were reduced (RA -290 +/- 30 vs. CIH -227 +/- 15 beats/min, P < 0.05); 2) HR responses to microinjection of NMDA into the left NA were reduced (RA -302 +/- 16 vs. CIH -238 +/- 27 beats/min, P < 0.05); and 3) the number of NMDAR1, AMPA GluR1, and AMPA GluR2/3-immunoreactive cells in the NA was reduced (P < 0.05). These results suggest that degeneration of NA neurons expressing GluRs contributes to impaired baroreflex control of HR in rats exposed to CIH.

Mechanism of myocardial ischemia with an anomalous left coronary artery from the right sinus of Valsalva.

OBJECTIVE An ectopic coronary artery that courses between the aortic root and the pulmonary trunk may lead to sudden cardiac death, especially in athletes. It has been speculated that during exercise, compression of the coronary artery between the great vessels may impair coronary blood flow and produce myocardial ischemia and fatal arrhythmia. However, this hypothesis cannot be tested in humans, and little experimental data exist to explain this phenomenon. To this end, in a calf with an anomalous left coronary artery that coursed from the right sinus of Valsalva between the great vessels, we assessed for myocardial ischemia during pharmacologically induced tachycardia and hypertension. METHODS We identified a juvenile male calf (103 kg) with an anomalous left coronary artery from the right sinus of Valsalva that coursed between the great vessels. Via thoracotomy, the animal was instrumented for hemodynamic measurements. Intravenous dobutamine increased heart rate and myocardial metabolic demands. Intravenous phenylephrine produced arterial hypertension and increased myocardial metabolic demands. Fluorescent-labeled microspheres were used to map regional myocardial blood flow, and hemodynamics were recorded during each condition. Massons trichrome staining for fibrosis, wheat-germ agglutinin staining for myocyte size, terminal deoxynucleotidyl transferase dUTP nick end-label staining for apoptosis, and isolectin-B4 staining for capillary density were performed. RESULTS For the first time, empiric data documented that an ectopic coronary artery produced myocardial ischemia during elevated myocardial metabolic demands. Left coronary artery resistance increased in a cardiac cycle-dependent pattern that was consistent with systolic compression between the great vessels. Increased cardiac fibrosis, myocyte hypertrophy, cardiac apoptosis, and capillary density indicated that regional ischemic, inflammatory-mediated myocardial remodeling was present. CONCLUSIONS These findings confirm the proposed mechanism of sudden death and support early surgical repair of coronary arteries that course between the aortic root and the pulmonary trunk.

Degeneration of vagal efferent axons and terminals in cardiac ganglia of aged rats

Baroreflex control of the heart rate is significantly reduced during aging. However, neural mechanisms that underlie such a functional reduction are not fully understood. We injected the tracer DiI into the left nucleus ambiguus (NA), then used confocal microscopy and a Neurolucida Digitization System to examine qualitatively and quantitatively vagal efferent projections to cardiac ganglia of young adult (5–6 months) and aged (24–25 months) rats (Sprague Dawley). Fluoro‐Gold was injected intraperitoneally to counterstain cardiac ganglionic principal neurons (PNs). In aged, as in young rats, NA axons projected to all cardiac ganglia and formed numerous basket endings around PNs in the hearts. However, significant structural changes were found in aged rats compared with young rats. Vagal efferent axons contained abnormally swollen axonal segments and exhibited reduced or even absent synaptic‐like terminals around PNs, such that the numbers of vagal fibers and basket endings around PNs were substantially reduced (P < 0.01). Furthermore, synaptic‐like varicose contacts of vagal cardiac axons with PNs were significantly reduced by approximately 50% (P < 0.01). These findings suggest that vagal efferents continue to maintain homeostatic control over the heart during aging. However, the marked morphological reorganization of vagal efferent axons and terminals in cardiac ganglia may represent the structural substrate for reduced vagal control of the heart rate and attenuated baroreflex function during aging. J. Comp. Neurol. 504:74–88, 2007.

Bovine model of chronic ischemic cardiomyopathy: implications for ventricular assist device research.

Ventricular assist devices (VADs) have emerged as a successful treatment option for advanced heart failure. The objective of this study was to develop a clinically relevant model of chronic ischemic cardiomyopathy to investigate functional, histological, and molecular changes during mechanical circulatory support. In calves (n = 17, 94 ± 7 kg), 90 μm microspheres were injected percutaneously into the left coronary artery. Serial echocardiography was performed weekly to evaluate cardiac function. Sixty days after coronary microembolization, a terminal study was performed via thoracotomy to measure hemodynamics. Regional myocardial and end-organ blood flows were quantified with 15-μm fluorescent-labeled microspheres. Myocardial fibrosis, myocyte size, and myocardial apoptosis were quantified with histological stains. Eleven animals survived coronary microembolization and exhibited clinical and statistically significant echocardiographic and hemodynamic signs of severe systolic dysfunction. Statistically significant decreases in regional myocardial blood flow and increases in myocardial fibrosis, myocyte size, total myocardial apoptosis, and cardiac myocyte-specific apoptosis were observed. End-organ hypoperfusion was observed. Coronary microembolization induced stable and reproducible chronic left ventricular failure in calves. The anatomical size and physiology of the bovine heart and thorax are appropriate to study novel interventions for the clinical management of heart failure. This model is an appropriate physiological substrate in which to test VAD and adjunctive biological therapies.

Mechanisms of triggered activity induction at the border zone of normal and abnormal cardiac tissue

Ionic mechanisms that may be involved in inducing triggered activations at the border zone (BZTAs) of normal and abnormal Purkinje fiber segments were investigated. In a two-chamber bath, fibers were divided into a normal segment and segment treated with ethylenediaminetetraacetic acid to stimulate electrophysiologic alterations 24 hours after infarct. Interventions to normal segments included 1.8 mM lidocaine (n = 10), 3 x 10(-4) mM tetrodotoxin (n = 5), 10(-3) mM aconitine (n = 4), 3 mM cesium chloride (n = 7), 10(-2) mM verapamil (n = 4), and 6-8 mM (n = 7) of K+. Ethylenediaminetetraacetic acid (3.3 mM) prolonged action potentials and induced low diastolic potentials in the normal segment border zone. Tetrodotoxin, lidocaine, and high K+ levels suppressed BZTAs; cesium chloride and aconitine increased BZTAs; and verapamil did not reduce BZTAs. The finding that BZTAs were not abolished by verapamil suggests that abnormal automaticity is not a mechanism. Apparently, BZTAs depend on the Na+ inward current activated by depolarization of the membrane secondary to depolarization of adjacent cells.

Nitric oxide mediates C5a-induced vasodilation in the small intestine.

Objective: This study was designed to investigate the microvascular responses of the small intestine to complement C5a and to define the role of nitric oxide in the C5a‐induced response.

In vivo effect of naftidrofuryl on 5-hydroxytryptamine-mediated constriction in rat peripheral microcirculation

Naftidrofuryl is commonly used in treatment of peripheral vascular disease. Its vasodilator action has been partly explained by its inhibitory effect of 5-HT2 receptors on peripheral arteries in vitro. The purpose of this study was to test in vivo whether naftidrofuryl selectively inhibits 5-hydroxytryptamine (5-HT)-mediated constriction of large arterioles in the peripheral microcirculation. This constriction appears to be 5-HT2 receptor-mediated. Three separate protocols were used to test the effects of naftidrofuryl: chronic injection (15 mg/kg, i.p., twice daily for 5-6 days; n = 7), acute intravenous (i.v.) infusion (15 mg/kg over 30 min; n = 7), or topical application (5 x 10(-8) M, n = 6; 5 x 10(-7) M, n = 5; 5 x 10(-6) M, n = 5; 10(-5) M, n = 7). Male Sprague-Dawley rats (145-185 g body weight) were anesthetized with sodium pentobarbital (50 mg/kg) and the cremaster muscle was prepared for intravital video microscopy. Diameter response of arterioles (70-120 microns) to increasing concentrations of locally applied 5-HT (10(-8)-10(-4) M) was assessed. In rats receiving no drug treatment, 5-HT caused vasoconstriction of arterioles beginning at 10(-6) M and reaching approximately 40% constriction at 10(-4) M. These vasoactive responses were not altered by chronic daily doses or an acute infusion of naftidrofuryl. 5-HT responses obtained with and without naftidrofuryl applied directly into the cremaster-bath also had little effect on the arteriole response at each of the four concentrations tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Using “Active Learning” Methods to Teach Physiology

The teaching faculty of the Department of Physiology and Biophysics at the University of Louisville School of Medicine have designed and implemented various “active learning” approaches within three different courses that teach physiology to medical and graduate students. The courses are: 1) medical physiology, 2) advanced physiology, and 3) integrated physiology. The unique aspect incorporated within these courses is that students prepare answers to pre-assigned questions or topics and then “teach” that topic to their peers. This pedagogical exercise encourages students to learn the material at a more comprehensive level and to speak the language of physiology. This paper presents the rationale and design for this type of approach within each course and provides feedback comments from both student and faculty participants. We also provide a list of some of the questions and topics used in two of these courses as a practical guide to readers who may want to implement similar approaches.