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The Autonomic Nervous System:
Its Study and The Pathogenesis of
Chagas' Cardioneuromyopathy*

Daniel Iosa, MD, PhD

C.P.D.I Córdoba, Argentina

INTRODUCTION
   American trypanosomiasis, or Chagas' disease, is one of the most frequent causes of congestive heart failure and sudden death in the world (1-4). Its pathogenesis is still a matter of debate, even though the disease was described by the Brazilian Carlos Chagas in 1909 (5,6). In 1913 this brilliant scientist already saw "a new frontier of nervous system pathology" (7), yet today the neuropathogenic nature of the disease continues to be debated, as well as the question of how it should be treated. This paper will hereafter refer to the "neurogenic theory" of Chagas' disease, as opposed to the competing "myogenic theory", which is espoused by many researchers.

   According to the myogenic theory, the primary damage to the cardiac muscle fibers is triggered by humoral or cell-mediated immune factors (8). However, this theory does not account for all known lesions or for their varied clinical manifestations, for Chagas' disease is a pleomorfhic clinical syndrome that produces symptoms and signs throughout virtually the entire organism (9).

   The neurogenic theory, on the other hand, can account for the chronic chagasic cardioneuromyopathy syndrome, as this will attrnpt to demonstrate.

   The first detailed description of neurological damage was published by Vianna in 1911 (10), in a study of the disease anatomic pathology. In 1919, Nováes, studying the peripheral nervous system, detected parasitized cells around the cutaneous nerves, near on even inside the perineurium (11). In 1924, Monckeberg (12) observed pronounced lesions in the autonomic ganglia and cardiac nerve fibers of experimentally infected dogs. Mazza and Jorg (13), in turn, found sympathetic ganglionitis in the sympathetic perirenal plexus in a naturally infected dog, with necrobiosis of the ganglionic cells.

   It is known that in the chronic phase of the disease, years after the acute infection, parasites circulating in the bloodstream or observable in tissue are reduced in number or totally disappear; at the same time, symptoms begin to appear or get worse. If this phenomenon is due to autoimmune processes triggered by the parasite or by the tissue damaged by the parasite's entry, the should be a direct relationship between the events in the acute stage and the lesions, signs, and symptoms in the chronic stage. This reasoning led Köberle to develop its "neurotoxin" theory in 1959. He (14-19) along with Alcántara (20), suggested that Chagas' disease is a neuropathy resulting from denervation caused by widespread destruction of parasympathetic neurons and nervous fibers in different areas - a theory that explained the occurrence of cardiopathy and megaviscera.

   Köberle neurological theory was based on two fundamental premises: first, the presence of parasympathetic neuron destruction accompananied by "relative" sympathetic hyperactivity, and second, the existence of a neurotoxic substance.

   The idea of parasympathetic neuron destruction came from anatomopathological studies that had demonstrated the existence of extensive areas of intrinsic denervation in various organs that presented alterations typical of Chagas' disease (16). Systematic counting of ganglionic cells in the intrinsic nervous system of all the organs studied (the bronchi, cerebellum, spinal cord, esophagus, colon, stomach, heart, etc.) showed unequivocal signs of parasympathetic denervation or neuron loss (16,18).

   The idea of a neurotoxin was inspired by the work of Vianna (10) and Monckeberg (12), who had described the destruction in canine models of nonparasitized cells subsequent to the rupture of nearby nests, as well as promounced lesions in the autonomic ganglia and cardiac nervous fibers. Such a neurotoxin would be found in the amastigotes as an endotoxin or enzyme capable of damaging all types of cells, but especially nerve cells. The resulting widespread damage would account for the multiform chagasic syndrome, with its digestive (aperistalsis, megaesophagus, megacolon), respiratory (megatrachea bronchiectasis), urinary (megaloureter), cardiac (denervation), and neurological (myelopathy, encephalopathy) components (20-22).However, the presence of a neurotoxin could never be demonstrated. In 1959 Musacchio and Meyer exposed nervous tissue cultures to a suspension of killed or degenerated. Trypanosoma cruzi cells and were unable to induce any lesion, leading them to conclude that there was in fact no neurotoxin (23). These findings collapsed on of the two pillars of Köberle's theory .

   The other pillar, the scenario of parasympathetic neuron loss in parallel with "relative sympathetic hyperactivity", was to be called into question by the anatomopathological findings of Alcántara himself, who in 1970 published his observations of denervation in intramural ganglia of the heart and cervicothoracic ganglia due to Chagas' disease (24). These observations were confirmed a year later when the same author found morphological and histochemical alterations in both parasympathetic cardiac neurons and sympathetic neurons of the cervicothoracic ganglia (25).

   This research lent support to the theory of neuronal and peripheral nerve lesions, but it showed that they occur in the sympathetic as well as the parasympathetic system. At this point, functional studies in human patients with chronic Chagas' disease became very important for providing further clarification (26).

   In 1922 Carlos Chagas used atropine to combat severe sinus bradycardia in his chronic chagasic patients, which shows that this condition had already been recognized (27). As will be seen, this observation was confirmed repeatedly (28-31).

   In 1968, Amorin et al. studied parasympathetic function by measuring bradycardia brought on by acutely elevated blood pressure and atropine induced tachycardia, which was altered as a result of parasympathetic dysfunction (32). In 1975 Marin Neto et al studied postural reflexes in chronic Chagas' disease patients and concluded that the abnormalities encountered in the tilting test could be due to sympathetic alterations or to interference in parasympathetic control (33). These authors obtained similar results in studies of tachycardia mechanisms through postural testing of normal and chagasic subjects (34).

THE AUTONOMIC NERVOUS SYSTEM IN THE CHRONIC CHAGAS' DISEASE: THEORY OF PROGRESSIVE NEUROTRANSMISSION BLOCKADE AND SUBSEQUENT DENERVATION
Arterial Hypotension in Chronic Chagasic Patients
   In 1973, Dr. Hugo Palmero became interested in for the fact that chronic chagasic patients frequently presented low blood pressure and slow pulse. It was known that Chagas' disease was associated with arterial hypotension, but there had been no statistical studies to support works this observation (35-38). The accepted explanation was that the low blood pressure resulted from reduced myocardial contractility and consequent failure of the heart's pumping action (38).

   To investigate the reasons and circumstances under which Chagas' disease produced arterial hypotension, Palmero, Caeiro and the present author (39) undertook a comparative study of systolic and diastolic pressure in chagasic patients and subjects from the general population of the same geographic area.

   A total of 115 chagasic patients were studied - approximately equal numbers of males and females, aged ± 13 years (mean ± standard deviation).
The patients were assigned to the following groups on the basis of their weight and height :
A). average weight ± 10% of ideal;
B). weight between 10% and 25% above ideal (overweight);
C). Weight more than 25% above ideal (obese).

   In addition, based on signs and symptoms, electrocardiogram (ECG) and chest X-ray, the patients were classified as follows:
I). without either heart failure or cardiomegaly (86);
II). with heart failure and cardiomegaly (29).

   The patients' blood pressure was measured according to the specifications of Hamilton et al (40) and W.H.O. (41,42).
The results may be summarized as follows:
A. Mean arterial pressure in the chagasic patients was fewer than in the general population, the differences being greater in women and for diastolic pressure.
B. there were no significant differences in arterial pressure between the chagasic patients with congestive heart failure (CHF) and those without.
C. Both the systolic and the diastolic pressure in chagasic patients without CHF were significantly lower than in the control group. This observation clearly demonstrated that heart failure could not be the only explanation for low blood pressure in chagasic patients.
D. In the chagasic patients, no direct correlation was seen between blood pressure and weight or age, as is observed in the general population, nor was there any correlation with sex (women over 35 normally have higher blood pressure than men). On the contrary, the older and more obese Chagasic patients often had much lower blood pressure than the average for the general population, regardless of whether or on they had CHF.
E. The prevalence of elevated systolic and diastolic blood pressure was lower in the patients of both sexes with chagas' disease. The percentages were as follows: (Table 1)

   Another discovery worthy of mention was that the pressure of the pulse (he/she differs between the systolic tensions and diastolic) in the chagásicos it spread to be bigger than in the general population, what moved away even more the possibility that the arterial hypotension would be due to heart inadequacy, since this diminishes the pressure of the pulse.

   These findings led to speculate that autonomic dysfunction may cause a type of ▀-blokc, similar to that produced by drugs (39). To clarify the findings and try to correlate them with the degree of clinical severity, further' studies were undertaken with the following five groups of subjects:
A) - Control group : healthy subjects, seronegative for Chagas' disease (negative Machado Guerreiro and inmunofluorescence tests), free of cardiovascular symptoms, with normal ECG and chest X-ray.
B) - Patients with nonchagasic congestive heart failure (NChCHF): patients with signs and symptoms of Grade III or IV CHF (per New York Heart Association diagnostic criteria (43), whether of coronary etiology or due to nonchagasic dilatated cardiomyopathy. This group did not include patients with valvular or recent or active acute myocardial ischemia. The findings from this group of patients were compared with those from the chagasic patients with CHF.
C) - CHAGAS I patients (ChI): seropositive asymptomatic individuals with normal ECG and normal X-rays.
D) - CHAGAS II patients (ChII): seropositive symptomatic persons with palpitations, abnormal ECG due to complete right bundle branch block, left anterior hemiblock, or arrhythmia, with normal chest- X-ray.
E) - CHAGAS III patients (EChIII): seropositive patients with signs and symptoms of CHF, abnormal ECG, and cardiomegaly visible in chest X-ray.

   The above classification scheme for chagasic patients has subsequently been the Argentine Council on Chagas' disease classification. Table 2 summarizes the characteristics of these five groups. Results of the studies in which they participated are reported in the following pages.

LOW HEART RATE IN CHRONIC CHAGASIC PATIENTS
   As noted above, sinus bradycardia had already been reported by Carlos Chagas as early as 1922 (27). Since chagasic patients often present marked sinus bradycardia, it was decided to conduct a study on the prevalence of this sign in chagasic patients by comparing basal heart rate in the various groups (28). In all, 222 chagasic patients were studied (80 in group ChI, 76 in ChII, and 66 in ChIII), as well as 50 healthy controls and 55 NChCHF patients.

   The subjects rested in a supine position for 10 minutes while their basal heart rate was recorded by an ECG machine that had previously has its velocity checked. Patients with atrioventricular block, frequent ventricular extrasystole, or fibrillation were excluded. The patients were not on any medication that might affect their involuntary behavior (▀-blockers or amiodarone), except for those in the two CHF groups -chagasic and nonchagasic- who were receiving cardiotonics - marking those two groups comparable. The results were as follows:
A - The chagasic subjects without CHF (ChI and ChII) had a slow heart rate, but the difference with respect to the control group was not statistically significant; however, many of the them showed a tendency toward sinus bradycardia.
B - The heart rate of subjects with chagasic CHF (ChIII) was significantly lower than that of subjects with NChCHF or control group members in all decades of life.

   These data differed substantially from the results obtained by Marín Neto et al (33), as well as those of Salgado et al (45). Both these teams had found higher heart rates, due to an apparent predominance or hyperactivity of the sympathetic nervous system.

   The study' data on bradycardia and arterial hypotension were likewise in disagreement with the theory of parasympathetic dysfunction with "relative" sympathetic hyperactivity proposed by Köberle, (14-22), since the hypothetical sympathetic hyperactivity would be expected to induce tachycardia and arterial hypertension. It seemed that in these patients the problem was not relative sympathetic hyperactivity but rather quite the opposite: sympathetic hypoactivity.

   The sinus node is often damaged in Chagas' disease, in many cases producing sick sinus syndrome, which necessitates pacemaker implantation (46,47).

   The sick sinus syndrome involves marked bradyarrhythmia. For it reason it is important to rule out the possibility of intrinsic damage to the sinus node before suspecting that intrinsic sympathetic flow to the node is reduced in asymptomatic chagasic patients who are found to have sinus bradycardia. In other cardiopathies, patients with CHF and bradyarrhythmia due to sick sinus syndrome are usually elderly, but in cases of Chagasic cardiopathy the patients already present marked bradyarrhythmia at an early age (8).

   A study of sinus node recovery time in the control group and in the chagasic subgroups that presented marked bradyarrhythmia rule out intrinsic damage in these patients and thus cleared the for the investigation of the autonomic nervous system (ANS)(48).

THE BARORECEPTOR REFLEX ARCH IN CHRONIC CHAGASIC PATIENTS: STUDY OF SYMPATHETIC-PARASYMPATHETIC REGULATION OF CARDIOVASCULAR FUNCTION
   The high prevalence of arterial hypotension in chronic chagasic patients regardless of the heart's pumping function, and of marked sinus bradycardia regardless of the patient's age, the degree of damage, or even concurrent CHF, as well as the lack of any alteration in sinus node recovery time in conditions under which it was logical to expect sinus, tachycardia, suggested the possibility of an ANS lesion, specifically, a lesion in the sympathetic nervous system. This subject had received little functional study in chronic Chagas' disease (29).

   Knowledge about normal and pathological function of the ANS has taken on considerable importance in recent years not only for researchers but also for practicing physicians and their assistants, who have seen a great increase in components of their therapeutic arsenal with direct and indirect effects on the ANS. The physician who treats a cardiovascular patient needs to understand the effects of selective and nonselective sympathetic ▀-blockers, with or without intrinsic sympathomimetic activity, as well as those of α (alpha)-receptor blockers and the more recent drugs that both α (alpha)-and ▀-receptors. Patients with obstructed airways are often prescribed ▀-2 receptor stimulants, and pharmaceutical laboratories are developing increasingly more specific drugs which stimulate or depress autonomic function.

   In many disease, ANS alterations are produced either as the result of the ayatem's own functional effort or because intrinsic anatomo-pathological lesions in the ANS generate the signs and symptoms of a pathological entity (49). Congestive heart failure, arterial hypertension, diabetic cardioneuropathy, and idiopathic postural hypotension are some of the cardiovascular disorders with ANS alterations that suggest the importance of taking a detailed look at the anatomic and functional characteristics of this system.

ANATOMY OF THE SYMPATHETIC NERVOUS SYSTEM
   The neurons from which the sympathetic nervous system originates are located in the lateral horn of the spinal cord (intermediolateral tract), starting at the C8 or T1 segment and extending to L2 or L3. The axons of these cells leaves the cord by way of the corresponding ventral nerve root and form a synapse with a peripheral ganglionic cell. The preganglionic fibers are unmyelinated. There are regulatory centers in the posterior region of the hypothalamus and in the cerebral cortex.

   The sympathetic ganglia are grouped into three systems (50): paravertebral, prevertebral, and terminal.

   The paravertebral ganglionic system consists of 22 pairs of interconnected ganglia flanking the spinal column. They form a nodular cord extending from the base of the skull to the coccyx and are known as the sympathetic trunk. Arising from the cervical portion of this chain are the fibers of the superior, middle, and inferior cardiac nerves, which together form the cardiac plexus.

   The prevertebral ganglionic system is located in the thorax, abdomen, and pelvis next to the aorta and its branches. The most important prevertebral ganglia are the celiac, the superior mesenteric, and the inferior mesenteric.

   The terminal ganglionic system is made up of small clusters of ganglion cells that are closely linked to the innervated organs, especially those of the pelvis, such as the rectum and the bladder.

   The nerves fiber that leaves the spinal cord and enters a paravertebral ganglion can a). synapse with a ganglionic cell of that ganglion, at the same cord level; b). Run up or down the sympathetic trunk until it forms a synapse with a paravertebral ganglionic cell located higher up or father down; or c). Continue on, without synapsing with any paravertebral ganglionic cell, until it reaches a prevertebral or terminal ganglion.

   The heart receives only postganglionic fibers, since the preganglionic fibers that emerge from the lower cervical and upper thoracic segments synapse in the stellate ganglion and the first three or four paravertebral ganglia. From there, the postganglionic fibers the right and left cardiac nerves travel toward the heart and form a plexus or very fine mesh which innervates both the heart muscle and the conduction system. The function of the ventricular myocardial fiber is influenced mainly by the sympathetic nervous system, which nervous system, which acts in situ through the release of norepinephrine, a neurotransmitter that mediates sympathetic activity through the ▀-1 receptors of the cardiac muscle and also through the α (alpha)-receptors of the coronary arteries and of some sinus node cells.

ANATOMY OF THE PARASYMPATHETIC NERVOUS SYSTEM
   The neurons that give rise to this system are found at three levels of the central nervous system (SNC): the mesencephalon (midbrain), the medulla ablongata (brain stem), and the sacral region of the spinal cord. The axons that leave the SNC are myelinated preganglionic fibers that synapse with ganglionic cells located in or very near the organs innervated by unmvelinated postganglionic fibers (51).

   The fibers from the mesencephalon originate in the Edinger-Westphal nucleus of the oculomotor nerve. The bulbar fibers travel outward along the tracts of the facial (VII), gliossopharyngeal (IX), and vagus (X) nerves.

   The sacral fibers, which originate in the ventral horn at the L2, L3, and L4 levels, and sometimes S1, form the pelvic nerve, which in turn becomes part of the pelvic plexus (51).

   Parasympathetic inervation of the heart comes from preganglionic fibers located inthe ambiguous nucleus, which reach the heart via the vagus nerve. At the heart, they synapse with ganglionic cells located in the atria and the base of the ventricles next to the conduction tissue; they then innervate, through the surface and deep cardiac plexuses of the atria, the nodules sinus and atrioventricular nodes, the initial portion of the ventricular conduction system, the coronary arteries, and certain muscle cells.

   The parasympathetic neurotransmitter in the heart is acetylcholine, to which the myocardial fiber is relatively insensitive (49).

PHYSIOLOGY OF THE AUTONOMIC NERVOUS SYSTEM
   The ANS innervates the heart and vessels through an integrated mechanism that involves both the parasympathetic and the sympathetic nervous systems, named by Ibrahim (52) the "baroreceptor reflex arc". This interconnected mechanism maintains stable cardiovascular tone through multiple instantaneous simulation-inhibition reactions, which regulate blood pressure level, heart rate, venous return, contractile status, and cardiac distensibility under varying conditions during daily activity (rest, exercise, stress, etc.).

   The baroreceptor reflex arc includes the baroreceptor corpuscles, IX and X cranial nerves, the bulbar vasomotor center, the sympathetic and parasympathetic efferent pathways, the blood vessels and their receptors, the somatic and autonomic afferent pathways, and the heart, including both its excitoconduction system and its muscle fibers (52).

   The baroreceptor corpuscles (from the Greek baros, meaning "weight") are not pressure receptors but rather small bodies which are sensitive to stretching, to changes in vascular diameter resulting from changes in intravascular pressure. Situated in the walls of the blood vessels and in the heart, they are concentrated most heavily in the breast carotid sinus and the aortic arch. They are also found in the right and left atria, in the walls of the superior and inferior venae cavae at their junction with the heart, in the pulmonary veins, and throughout the pulmonary circulation (figure 1) (53). The IX and X cranial nerves constitute the afferent pathway for the baroreceptor corpuscles and are responsible for conducting the information about intravascular pressure levels which is carried by these bodies, with each heartbeat, to the bulbar vasomotor center.

Figure 1. Anatomy of the autonomic nervous system.
BVC = bulbar vasomotor center; IX and X = afferent pathway of IX and X cranial nerves; b = baroreceptor corpuscles; SEP = sympathetic efferent pathway; sc = spinal cord; VEP = vagal efferent pathway; g = sympathetic ganglia.

   Functionally, this vasomotor center has two distinct areas: a sympathoexcitatory cardioaccelerator area, and a sympathoinhibitory cardiodepressor area (figure 1). Depending on the system's needs, these areas, through their corresponding efferent pathways, produce either tachycardia and peripheral vasoconstriction or bradycardia and vasodilation. The vagus (X cranial) nerve is the efferent pathway that goes to the sinus node, where its stimulation generates bradycardia. Its inhibition, on the other hand, generates tachycardia by allowing the sympathetic efferent fibers to induce increased heart rate and vasoconstriction. Inhibition of these latter effects produces peripheral vasodilation.

   An example will illustrate the integration function of the baroreceptor reflex arc: a sharp fall in arterial pressure, due to hypovolemia produced by acute hemorrhage, stimulates the baroreceptors, which transmit the information to the bulbar vasomotor center through the IX and X afferent nerves. The vasomotor center receives this message along with other information transmitted to it by its own pressure receptors. Through its stimulatory and cardioaccelerator area, it produces a more or less immediate reaction to the fall in blood pressure - a reaction sent through the sympathetic efferent pathway that will trigger an increase in blood pressure by means of venous and peripheral arterial vasoconstriction, tachycardia, and increased inotropism. In This way, the baroreceptor reflex arc helps to restore normal blood pressure levels. The same thing happens in reverse in the event of an increase in intra-arterial pressure, for example following an injection of phenylephrine (31).

STUDY OF THE FUNCTIONAL INTEGRITY OF THE AUTONOMIC NERVOUS SYSTEM IN CARDIOVASCULAR REGULATION
   A thorough functional study of the ANS includes determination of the integrity of the baroreceptor reflex arc; a functional study of the bulbar vasomotor center, the sympathetic efferent pathway, the arterial smooth muscle receptor response, and the vagal afferent pathways; verification of norepinephrine storage in the sympathetic nerve terminals; and determination of plasma catecholamine levels. These aspects are examined in detail in the eight sections that follow.

DETERMINATION OF THE INTEGRITY OF THE BARORECEPTOR REFLEX ARC
   This study is accomplished by examining the effect of Valsalva's maneuver and the tilting or postural test on blood pressure and heart rate. The response to Valsalva's maneuver reveals whether the reflex arc is normal or pathological (54). A pathological state would be due to a sympathetic alteration when there is no tachycardia in the compression phase, and would be due to a parasympathetic alteration when bradycardia is absent in the decompression or overshooting phase (6) (figure 2).

Figure 2. Valsalva's maneuver. Continuous monitoring of electrocardiogram (ECG) and intra-arterial pressure (BP) in mmHg during the four phases.
S = start of compression phase (straining phase); R = start of decompression phase (releasing phase); ** = initial and final overshooting.

   The tilting test consist of having a subject move from a supine to an upright position either passively, by means of a swiwel-mounted table (55), or pressure and heart rate with little or on decrease in systolic pressure (figure 3). The production of these changes involves the aortic and carotid baroreceptors as well the atrial receptors, the afferent pathways of these corpuscles, the bulbar vasomotor center, and the sympathetic efferent pathway (49, 55).

Figure 3. Passive tilting test. Increase in diastolic pressure (BP) is seen in the control group (CG); chagasic patients in groups ChI, ChII and ChIII; and patients with nonchagasic congestive heart failure (NChCHF) at 1, 2, 3, 4 and 5 minutes after the tilting test (adapted from reference 29).

FUNCTIONAL STUDY OF THE BULBAR VASOMOTOR CENTER
   Voluntary hyperventilation for 15 seconds induces a fall in systolic and diastolic blood pressure as well as a reflex increase in heart rate (figure 4) (57). Hyperventilation causes a reduction in cerebral Pco2, which results in cerebral vasoconstriction and consequent hypertension at the bulbar level. The sympathetic inhibition area of the bulbar vasomotor center then sends impulses along its efferent pathway which cause peripheral vasodilation (57,58).

Verification of a fall in blood pressure triggered by hyperventilation is a sign that the bulbar vasomotor center, its efferent pathway, and its terminal organ are functioning normally (57).

Figure 4. Hyperventilation maneuver. Above, electrocardiogram reading; below, continuous intra-arterial pressure reading (mmHg). I = initial, F = end of the maneuver. In a normal subject, decreases in systolic and diastolic pressure and an increase in heart rate are observed at the end of hyperventilation, followed by immediate recovery from the maneuver.

FUNCTIONAL STUDY OF THE SYMPATHETIC EFFERENT PATHWAY
   This pathway can be studied by means of the following tests:
A. Cold pressor test: Submersion of the hand in water to 4 ºC produces painful stimuli which are transported via the lateral spinothalamic tracts. If the sympathetic pathway is intact, the efferent response induces a rise in both systolic and diastolic pressure (52). However, this rise does not take place in subjects with autonomic receptor blockade, whereas the response is exaggerated in those suffering from denervation (figure 5) (52).
B. Arithmetic test: Mental stress induces a rise in blood pressure. The test is conducted by asking the subject to successively subtract a certain number (8, for example) from 100 and to give the results aloud : 92, 84, 76. . .etc. (52).
C. Sweating test : Exposure of the subject to an warm environment produces a sweating reflex mediated by the sympathetic efferent fibers. If sweating does not occur, it is then necessary to verify by direct glandular electrostimulation (iontophoresis) that the sweat glands are capable of proper functioning. If they are, it may be concluded that the effect was the result of an alteration in the sympathetic afferent pathway.

Figure 5. Cold pressor test. Basal systolic and diastolic pressures (B) and readings subsequent to immersion in five groups of subjects: CG = control group; ChI, ChII and ChIII = Chagas' disease patients, chronic stages I, II and III, respectively; NChCHF = nonchagasic congestive heart failure. Number of subjects in each group indicated in parentheses.

FUNCTIONAL STUDY OF ARTERIAL SMOOTH MUSCLE RECEPTOR RESPONSE
   Intravenous administration of phenylephrine (25-50 ug) is used to measure the receptor response in arterial smooth muscle. If the vessels´ receptors are functioning normally, this α (alpha)-agonist produces an increase in systemic arterial pressure through vasoconstriction (31,52).

FUNCTIONAL STUDY OF THE VAGAL AFFERENT PATHWAY
   This pathway is also studies by means of an intravenous injection of phenylephrine , which induces a rise in blood pressure. If the afferent and afferent pathways are functioning normally, the increase in pressure will produce a reflex bradycardia. The fibers being tested are those in the baroreceptor reflex arc (IX and X cranial nerves) (59).

FUNCTIONAL STUDY OF THE VAGAL EFFERENT PATHWAY
   As explained above, both the afferent and the efferent pathways may be studied by observing whether a rise in blood pressure produces bradycardia. This rise in arterial pressure may be induced by a injection of phenylephrine or in the decompression phase of Valsalva´s maneuver (Figure 1 and 2) (59).

   Another way to study the vagal afferent pathway is intravenous injection of atropine (0,04 mg/kg), which suppresses vagal action. If the vagus was functioning correctly prior to the injection, there will be a 30 to 40% increase in heart rate above the basal level in normal subjects.

VERIFICATION OF NOREPINEPHRINE STORAGE IN THE SYMPATHETIC NERVE TERMINALS: TYRAMINE TEST
   Normally, norepinephrine (noradrenaline) is synthesized in the terminals of the sympathetic nerve fibers and then released from vesicles to transmit a nerve impulse to the corresponding receptor. Tyramine is a sympathomimetic amine that indirectly produces a pressor effect by causing the norepinephrine accumulated in the neurovascular terminal to be released (52). If the receptor is blocked, even if there are normal amounts of norepinephrine in the nerve terminals, and injection of tyramine does not induce an increase in arterial pressure.

DETERMINATION OF PLASMA CATECHOLAMINE LEVELS
   Sympathetic neuronal activity releases epinephrine and norepinephrine, which ar used in neurotransmission, metabolized, and recaptured by the nerve terminal. Measurable concentrations circulate in the blood (3,36). Part of the circulating norapinephrine is effectively recaptured in distant organs and part of it is eliminated through the urine. Its concentration in 24-hour urine samples is also a reliable indication of sympathetic activity (3). It might be supposed that norepinephrine concentration depends much more on adrenal gland activity than neurotransmission, but this is not the case, since the concentration remains unaltered following bilateral adrenalectomy (61).

   One way to avoid injection tyramine would be to measure plasma norepinephrine concentration immediately before and after the tilting test. This test produces a sympathetic stimulus that causes the instantaneous release of norepinephrineto the terminal organ, generating a rise in diastolic blood pressure, heart rate, and plasma concentration of the neurotransmitter. If these events happen normally, it can be concluded that the neuron, the neurofibril, and the terminal organ are all intact (3).

THE BARORECEPTOR REFLEX ARC IN CHRONIC CHAGAS' DISEASE
Assessment by the Tilting Test
   The change from supine to upright position traps peripheral venous blood and reduces venous return to the heart. The result is a lowering of the filling pressure of the right and left ventricle and a drop in minute volume (62). In normal subjects, mean blood pressure levels are maintained through an increase in diastolic pressure and heart rate, with little or no change in systolic pressure (55). These changes are brought about by the aortic and carotid baroreceptors as a response to the initial fall in arterial pressure, which triggers a reflex sympathetic stimulation in the vessels and the heart (63). The efferent pathway of this reflex involves adrenergic fibers that release norepinephrine in the vessel wall (64).

   The purpose of the study described below was to investigate whether or not the baroreceptor reflex arc was damaged in chronic Chagas' disease, with special focus on sympathetic activity (i.e., increase diastolic pressure and heart rate). The tilting test had already been used by other investigators in the study and demonstration of parasympathetic alterations (33). It had been established that CHF per se can induce alterations in sympathetic and parasympathetic function, (65-67) which could account for some or all of the autonomic dysfunction described in Chagas' disease (29). In order to further Clarify this point, this study compared chagasic patients with and without cardiopathy with normal controls and with patients who had nonchagasic CHF (29). A total of 34 chagasic patients were studied: 10 of them without cardiopathy (ChI), 10 of them with cardiopathy but without cardiomegaly (ChII), and 14 with chagasic heart failure and cardiomegaly (ChIII). There were 10 persons in the control group and 10 in the group of nonchagasic patients with CHF.

   The tilting test was done passively, with a foot support for moving the subject into an upright position. The following results were obtained :
A). All the chagasic patients, regardless of whether or not they had CHF, responded to the tilting test with a significantly lower increase in diastolic pressure than did the controls.
B). The heart rate of chagasic patients with heart failure was significantly lower than that of either the control group or those with nonchagasic CHF;
C). During the 5 minutes of the tilting test the chagasic patients - both those with and those without heart failure - experienced changes in diastolic pressure that were similar to the changes in nonchagasic patients with CHF (figures 3) (29).

   Since postural stress failed to induce vasoconstriction and a rise in diastolic pressure in the chagasic patients, even those without demonstrable cardiopathy, it could be concluded that these patients had an altered sympathetic response to such stress.

   This finding ruled out the possibility that CHF per se could account for their autonomic dysfunction, which therefore would be independent of chagasic etiology. The study also corroborated a very important difference between the dysautonomia of chagasic and nonchagasic CHF patients: while both groups had a similar diastolic response, the latter group could be clearly distinguished from the chagasic patients in that they presented sinus tachycardia (29).

ASSESSMENT BY VALSALVA'S MANEUVER
   Valsalva's maneuver basically consists of recording basal HR and blood pressure during normal breathing followed by a single deep breath (phase 1), forced exhalation with the glottis closed for 15 seconds (compression, phase 2), a forceful exhalation (phase 3), and normal breathing again (decompression or overshooting, phase 4) (figure 2) (68). Phase 2 (compression) and phase 4 (decompression or overshooting) are the most important ones in the functional assessment of the ANS (6).

   The fall in blood pressure accompanied by reflex tachycardia at the end of phase 2 is used to evaluate sympathetic activity. Phase 4 causes an abrupt rise in blood pressure with reactive bradycardia, from which it can be determined whether or not the parasympathetic pathway has been affected. This latter phase is also used to measure the baroreceptor index, since the curve of rising blood pressure and consequent reduction in pulse is almost identical to the effect achieved by the injection of phenylephrine (31,59). Since parasympathetic alteration had been previously demonstrated and discussed in the literature (69,70), studies were done in which the results from the compression ("sympathetic") and decompression parasympathetic") phases of Valsalva´s maneuver in the groups of chronic chagasic patients were compared with those in the healthy controls.

   In the compression phase, all the chagasic subgroups had less pronounced tachycardia than the normal subjects. The percentage variations of the R-R interval from the basal level (mean ± SD) were as follows: controls, -27,0 ± 4,3; ChI and ChII, 14,0 ± 6,9; ChIII, - 11,6 ± 18.

   In the decompression phase, all the chagasic subgroups had less pronounced bradycardia than the normal subjects. The percentage variations of the R-R interval from the basal level were as follows (mean ± SD): control group, 44,6 ± 38,0; ChI and ChII, 17,0 ± 22,1; ChIII, 1,7 ± 5,4.

   On the basis of these results it was concluded that both the sympathetic and parasympathetic phases of Valsalva's maneuver are altered in chronic chagasic patients (6); that these alterations are already apparent in early Chagas' disease, in patients without demonstrable cardiopathy (ChI); (6) and, therefore, that CHF is not the cause of these alterations but instead perhaps the effect, since even the patients in group ChI (asymptomatic with normal ECG and chest X-ray) showed these alterations. The conclusions reached here do not agree with those of other investigators, who have suggested that the denervation observed in Chagas' disease is the consequence of abnormal ventricular function (70).

BULBAR VASOMOTOR CENTER FUNCTION: ASSESSMENT BY THE HYPERVENTILATION TEST
   The reflex induced by hyperventilation does not involve either the previous maneuvers. It has been established that this reflex tests the functioning of the bulbar vasomotor center, the sympathetic efferent pathway, and the terminal organ - in other words, exclusively sympathetic efferent function (figures 1 and 4) (57).

   In normal subjects, systolic and diastolic pressure drops by about 30 mmHg at the end of hyperventilation and then quickly returns to basal levels (50,52,57,58). Patients with autonomic dysfunction, on the other hand, show a steady fall in arterial pressure, and their return to basal levels may take quite a few seconds or even minutes (57).

   To obtain precise information on bulbar vasomotor center function in chronic chagasic patients, the hyperventilation maneuver was performed on 8 subjects from the control group (mean age ± SD; 32 ± 8,3 years) and 33 chagasic patients from groups ChI, ChII and ChIII (39,5 ± 7,4 years old). The maneuver was performed in the usual way, following canalization of the humeral artery to obtain uninterrupted readings of intra-arterial pressure and heart rate (figures 4) (57,58).

The results were as follows:
A). In the three chagasic subgroups the fall in arterial pressure immediately following hyperventilation was significantly less than in the control group for both systolic and diastolic pressure (figures 6 and 7).
B). Those in the control group recovered their basal values within 8 seconds, whereas those in the three chagasic groups took more than 20 seconds, demonstrating that they were unable to regain their normal blood pressure quickly (Figures 6 and 7).
C). There were no statistically significant differences in blood pressure response between the three chagasic groups.
D). The ChIII group had a significantly lower heart rate than the controls.
It was therefore concluded that the lesion in the baroreceptor reflex arc cannot be localized exclusively in the afferent pathway or in the parasympathetic system. The first possibility was ruled out because the maneuver does not involve the afferent pathway, and the second, because the test measures sympathetic function only (57,58).

Figure 6. Hyperventilation maneuver. Fall in systolic pressure inmediately after completion of the maneuver and subsequent recovery 8 seconds later. D BP = change in blood pressure; CG = control group; ChI, ChII and ChIII = Chagas'disease patients, chronic stages I, II and III, respectively; *** = statistically significant (adapted from reference 58).

Figure 7. Hyperventilation maneuver. Fall in diastolic pressure inmediately after completion if the maneuver and subsequent recovery 8 second later.
D BP = change in blood pressure; CG = control group; ChI, ChII and ChIII = Chagas'disease patients, chronic stages I, II and III respectively; *** = statistically significant (adapted from reference 58)

The finding that all three chagasic groups responded abnormally also ruled out CHF per se as the cause of this dysfunction.

The results of the hyperventilation test showed that chronic Chagas' disease is capable of inducing alterations in the sympathetic nervous system even in the absence of demonstrable cardiac disease. The lesion must be located in the bulbar vasomotor center, the sympathetic efferent pathway, or the terminal organ. These findings at least partially explained the sinus bradycardia and arterial hypotension that have described in chronic chagasic patients (28,39).

THE SYMPATHETIC EFFERENT PATHWAY: EVALUATION WITH THE COLD PRESSOR TEST
The hyperventilation test demonstrated an alteration in the sympathetic efferent pathway in chronic Chagas' disease, and the simple cold pressor test, in turn, made it possible to characterize the sympathetic lesion more specifically (6). In studies of the groups already mentioned, the following results were obtained:
a). A normal increase in systolic and diastolic blood pressure was seen in the control group.
b). ChI and ChII patients showed a somewhat lower response compared with the controls, but the difference was not statistically significant (figure 5);
c). A significantly increase occurred in CHIII patients versus all the others - the ChI, ChII and NChCHF patients and the controls (figure 5).

These results suggested that there might be a neurotransmitter blockade in the ChI and ChII patients and that the ChIII group might be having, in accordance with Cannon´s law , some form of hyperactivity due to denervation (6).

STUDY OF THE BARORECEPTOR INDEX BY INJECTION OF PHENYLEPHRINE
To analyze baroreceptor functional status in Chronic Chagas' disease, studies were conducted on 32 chagasic patients from the three groups mentioned, aged 39 ± 7,2 years (mean± SD), plus 8 healthy controls, aged 27,3 ± 11,7 years. With the subject lying in a supine position, the humeral artery was canalized, basal heart rate and intra-arterial pressure were recorded, and phenylephrine (50 ug) was injected intravenously. Within a few seconds there was a rise in blood pressure witch each beat which in turn induced varying degrees of bradycardia as an efferent response by the baroreceptors that had been stimulated (31). In the regression model, with X representing blood pressure and representing heart rate, the regression coefficient was considered to be an index of baroreceptor sensibility. The results were as follows:
A). The average baroreceptor sensitivity index was significantly higher in the controls than in any of the three groups of chagasic patients (ChI, ChII, or ChIII).
B). The average baroreceptor sensibility index fell in direct proportion to the clinical severity of the disease in the three groups of chagasic patients. Thus it was possible to confirm that the baroreceptors are altered in chronic Chagas' disease, even in cases without any evidence of cardiopathy, and that alteration becomes more marked with increasing clinical severity of the disease.

The tyramine test has never been performed by is in Chagas' disease as it was replaced by the test described below.

MEASUREMENT OF PLASMA NOREPINEPHRINE DURING THE POSTURAL TEST
Plasma norepinephrine concentration is a very sensitive index of neurovascular activity, both in normal functional states and in dysautonomias (72). Nonchagasic CHF patients exhibit sympathetic hyperactivity, and their norepinephrine concentrations both at rest and during exercise are significantly higher than those of normal subjects (73). This sympathetic hyperactivity leads to a series of clinical manifestations associated with heart failure : tachyarrhythmia, increased sweating, cold skin, pallor, anxiety, edema, oliguria and hypernatremia (74). Previous studies have shown that chronic Chagas'; patients, on the other hand, have an ANS alteration that could lead to clinical manifestations that differ from those of other CHF patients - bradyarrhythmia, asthenia, low blood pressure, reduced sweating, less anxiety, etc. (9) - which would suggest diminished sympathetic function in chronic chagasic cardiopathy.

Accordingly, measurements were made of plasma norepinephrine concentrations (75) in normal subjects and patients with varying degrees of Chagas' disease using a radioenzymatic assay. The concentrations found were then correlated with simultaneous changes in diastolic pressure and heart rate measured during the active postural test (3) (figure 8). The purpose was to see whether the sympathetic alteration was due to (1) blockade of neurovascular α (alpha)-receptors, in which case levels of norepinephrine would be normal or elevated but little or no increase in diastolic pressure would occur when the subject stood upright (figure 8), or (2) blockade of the neurovascular receptors with partial denervation, in which case levels of norepinephrine would be lower than expected and there would be no increase or possibly a decrease in diastolic pressure in the upright position.
The results were as follows:
a). In all groups, systolic pressure changed only slightly or not al all in response to the upright position.
b). Diastolic pressure, on the other hand, increased normally in the controls, did not increase in the ChI and ChII patients, and fell in the ChIII group (figure 8);
c). Heart rate rose in the controls and in the ChI and ChII patients but did not increase in the ChIII patients;
d). The plasma norepinephrine levels in the control group (in supine position, 388 ± 46 ng/l (mean + SE); in upright position, 585 ± 64 ng/l) were similar to those obtained in other studies, which validated the methodology used (72) (figure 8);
e). Plasma norepinephrine increased in the tilting test, the highest levels being observed in the ChII group;
f). Plasma norepinephrine reached significantly higher levels in the NChCHF patients (870 ± ng/l) than in the ChIII patients (359 ± ng/l) (figure 8).

On the basis of these results it was concluded that in the initial stage of Chronic Chagas' disease there is partial blockade of α (alpha)-1 and α (alpha)-2 receptors similar to that observed in some patients treated with blockers. This blockade prevents an increase in diastolic pressure when the patient stands up. In this stage (ChI), the plasma norepinephrine levels are normal (figure 8), whereas in more advanced stages (ChII), when the receptors experience a greater degree of blockade, plasma norepinephrine levels tend to be high, which correlates with the appearance of arrhythmias (3,37). In the late or most serious stages of the disease (ChIII), the patients suffer from CHF, but their plasma norepinephrine levels are not as high as those of patients with nonchagasic CHF, who have sympathetic hyperactivity; on the contrary, their levels are similar to those of subjects without heart failure. Nevertheless, the ChIII patients have a more abnormal blood pressure response in the tilting test, which is typical of a state of partial denervation in a previously blocked neurovascular receptor (3) (figure 8).

Figure 8. Simultaneous measurement of variation in diastolic pressure (mmHg, blocks) and plasma concentration of norepinephrine (ng/L, asterisks) in the active postural test. Control group (CG); Chagas'disease patients, chronic stages I, II and III (ChI, ChII and ChIII, respectively); and patients with nonchagasic congestive heart failure (NChCHF). The contrast between the normal response in CG subjects and the pathological responses in the chagasic and nonchagasic patients with congestive heart failure can be clearly seen.

GENERAL DISCUSSION AND CONCLUSIONS
   The foregoing findings with respect to baroreceptor reflex arc function in Chronic Chagas' disease suggest the following scenario for the pathogenesis of chagasic cardioneuropathy:
T. cruzi enters the organism and, by immune mechanisms yet to be clarified, produces a subtance we will call "substance I", which has distinctive immunologic properties. During the indeterminant period, or latent years, between the acute phase of the disease and the chronic phase when lesions appear, this "substance I" - through such diverse mechanisms as lymphocyte-mediated reactions; antilaminin or anti-basal-membrane antibodies; antineuron antibodies; antigen-antibody deposits in the vascular endothelium with microvascular lesions muscle fibers, or nerve terminals; peripheral antinerve antibodies; antiganglioside antibodies; and cross-reactions between host cells ant T. Cruzi (6,76-80) - induces the following:
a). Progressive blockade of the neuromuscular and neurovascular receptors in ChI and ChII patients;
b) Partial denervation, in addition to the preexisting blockade, in chronic chagasic patients with CHF (ChIII).
The blockade-denervation sequence of the clinical stages of the disease may be explained as follows;

   1). Patients with level I Chagas' disease (ChI) show no signs of cardiopathy in their ECG. Both this test and the chest X-ray are normal (cardiothoracic index lower than 0,50) (45,58). They have no signs or symptoms of CHF or any other cardiopathy. However, upon close questioning they complain of weakness, muscular asthenia, and morning fatigue. Many of them, even at very young ages, have gallstones, and they also tend to have low blood pressure and a slow pulse (9,81). Blockade of the receptors is slow and progressive, so that in the initial phase of the disease the patients have few symptoms. However, because of the neuromuscular and neurovascular blockade in both the sympathetic and parasympathetic systems, systolic and diastolic blood pressure in these patients is lower than in the general population. They also experience sinus bradycardia, do not undergo a rise in diastolic pressure in the tilting test, show less tachycardia and bradycardia than normal subjects during the compression and decompression phases of Valsalva´s maneuver, have reduced baroreceptor sensitivity and a lower index, and show diminished reactivity in the hyperventilation, coughing reflex, and cold pressor tests (3,9,58,71,82). These changes must not be due to a decrease in minute volume, since this group of patients is known to have a normal ejection fraction, as measured by radiocardiography and echocardiography (83,84). Because of the receptor blockade, these ChI patients are incapable of responding with an elevation of diastolic pressure to the sympathetic stimulus induced by the tilting test, despite the fact that they have normal plasma norepinephrine levels. Indeed, their diastolic pressure not only fails to rise but actually goes down (-1,1 mmHg with 595 ng/L norepinephrine in the ChI group versus - 6,4 mmHg with 578 ng/L norepinephrine in the control group) (3,6) (it figure 8).

   2). In patients with level II Chagas';disease (ChII), the nerve receptor blockade intensifies, as can be seen from the abnormal results in tests that involve the baroreceptor reflex arc. These patients often suffer from palpitations, fatigue, muscular asthenia, gallstones, and digestive symptoms, as well as dysautonomic symptoms such as nausea and postural hypotension (6). The abnormalities described in the ChI group in autonomic tests are more marked or more diverse in the ChII patients. The neurotransmitter blockade invariably produces elevated plasma concentrations of the corresponding neurotransmitter, and these patients present significantly higher norepinephrine levels compared with both the control group and the ChI patients (3) (figure 8). The blockade prevents the neurotransmitter from acting effectively on the receptor; as a result, diastolic pressure falls to rise in the tilting test despite the fact that the patient has high plasma concentrations of norepinephrine (figure 8). These concentrations account in part for the arrhythmias often seen in such persons (3,73).

   3). The patients with level III Chagas' disease (ChIII) are the most symptomatic. All the signs and symptoms mentioned so far become more marked and more frequent in this group and are compounded by signs and symptoms of CHF with generalized cardiomegaly (9). The intensity of the receptor blockade is even more evident in the tests that measure autonomic dysfunction.

   In addition, these patients also exhibit the following signs that are typical of partial denervation:
a). Plasma norepinephrine, which is usually quite high in nonchagasic CHF, is not elevated in ChIII patients; instead, concentrations are actually lower than those in group ChII, making for a false normalization effect (3) (figure 8). The tilting test gives the results that might be expected in light of low levels of plasma norepinephrine and blocked receptors: diastolic pressure falls by almost 5 mmHg and the heart rate cannot accelerate as it does in the normal condition or in nonchagasic heart failure (figure 8).
b). these patients are hyperreactive to the cold pressor test, which is a typical response of denervated organs (9) (it figure 5).

   Because of the sympathetic hypoactivity that occurs in ChIII patients, chagasic CHF is unique among the dilatated myocardiopathies, which typically cause tachycardia, sweating, pale and cold skin, and elevated levels of plasma norepinephrine. In contrast, CHF in chronic Chagas' disease is characterized by an absence of anxiety and sweating, bradycardia, and low plasma concentrations of norepinephrine (3). Thus there is a need to clearly differentiate between chagasic cardioneuromyopathy heart failure, in which the basic problem is neuromuscular-microvascular, and the other myocardiopathies, in which the problem stems from the myocardial fiber.

   Sympathetic hypoactivity could very well account for the high incidence of sudden death in this group of chagasic patients, since a close correlation has been established between low sympathetic activity and sudden death (85).

   In light of the results described here, as well as the findings of other authors (8,86) it seems logical that the cardiovascular disorder found in Chagas' disease should be called a "cardioneuromyopathy" rather than a "cardiomyopathy". The term chagasic cardioneuromyopathy is justified not only in light of the neuromuscular pathogenesis of the condition but also because of the importance of clearly differentiating between the chagasic disorder and the cardiomyopathies.

RECENT STUDIES THAT SUPPORT THE PROPOSED NEUROGENIC THEORY
   Following completion of the study of the baroreceptor reflex arc, an attempt was made to substantiate the findings by other methods that also assess sympathetic and parasympathetic function and, at the same time, to gain a better understanding of the neuroimmune mechanisms at work in pathogenesis at different stages of chronic Chagas' disease (ChI, ChII, and ChIII). With these goals in mind, studies of total pharmacological denervation were carried out; heart rate variability was examined by power spectral analysis; and anti-T cruzi, anti-sciatic nerve, and antiganglioside antibodies were investigated.

TOTAL PHARMACOLOGICAL DENERVATION (COMBINED AUTONOMIC BLOCKADE WITH ATROPINE AND PROPRANOLOL) IN CHRONIC CHAGASIC PATIENTS
   These studies were conducted following the José and Taylor protocol (87). Atropine (0,04 mg/Kg) was administered intravenously, followed minutes later by propranolol (0,2 mg/Kg). The atropine inhibits parasympathetic activity and the propranolol blocks the ▀-1 and ▀-2 sympathetic receptors. The subsequently measured heart rate is known as the observed intrinsic heart rate (OIHR). Since HR normally tends to decline with age, it is necessary to correct this factor with the predicted intrinsic heart rate for age (PIHR), which is calculated using José's formula [117,5 - (0,53 x age)] (88). The OIHR is considered to be just as effective as the sinus node recovery time for the assessment of sinus node function (89-90).

   Thirty-seven chronic chagasic patients were studied, 19 males and 18 females, aged 41 ± 12 years (mean ± SD); 14 were from group ChI, 14 from ChII, and 9 from ChIII. In addition, 6 healthy volunteers, ages 38 ± 10 years, served as controls. Statistical analysis was performed using Student´s test for unpaired samples and one-way analysis of variance (ANOVA).
The results may be summarized as follows:
1). In the ChIII patients, basal heart rates were significantly lower than in the controls or the ChI and ChII groups. This result is consistent with previous findings of bradyarrhythmia in chronic chagasic CHF.
2). In response to injection of atropine, the three chagasic groups showed a lower increase in heart rate than the control group. In the controls the average change was + 68, whereas it was + 45 in ChI, + 45 in ChII, and + 32 in ChIII (P <0,001, P <0,01 and P <0,001, respectively) (figures 9).
3). OIHR was measured following the injection of propranolol. The changes relative to basal heart rate were smaller in the three chagasic groups (ChI, + 17; ChII, +17; ChIII +5) than in the controls (+29) (P <0,05, P <0,02 and P <0,001, respectively) (figure 9).
4). In the control group the difference between the OIHR (104 + 14 beats /min.) and the PHIR (97 + 5,2) was not statistically significant, which validated the methodology used.
The results suggested the following : (91,92)
A. - A reduction in vagal activity was indicated by the reduced response to atropine in the three chagasic groups. The lowest response to atropine occurred in the patients with CHF, However, CHF only partially accounts for this altered response, (65) since the ChI and ChII groups, in which there is no cardiac insufficiency, had a similar, albeit less pronounced, response (figure 9). This finding was suggestive of a progressive lesion.
B. - A lowered response to propranolol was seen in the three groups of patient. This result was consistent with previous pathological and physiological observations demonstrating the presence of sympathetic alteration in chronic Chagas' disease.

Figure 9. Increase in heart rate under conditions of total combined pharmacological autonomic blockade with atropine and with atropine plus propranolol (OIHR = observed intrinsic heart rate). The progression of increased heart rate can be clearly seen in both the control group (CG) and the chagasic patients (ChI, ChII and ChIII).

ANALYSIS OF HEART RATE VARIABILITY BY POWER SPECTRAL ANALYSIS IN CHRONIC CHAGASIC PATIENTS
   Power Spectral analysis was used to investigate chagasic cardioneuromyopathy (93,93) because several studies had indicated that it was a very sensitive method for evaluating the autonomic alterations that occur in CHF, diabetic cardioneuromyopathy, acute myocardial infarction, and essential hypertension (95-98).

   The study included 31 subjects: 12 controls, 7 in group ChI, and 12 in group ChII. The age, blood pressure, and weight of all the subjects were similar (94). Each patient was connected to a two-channels Holter monitor (Del Mar Avionics, Irvine, California) and monitored continuously under the following conditions :
a). Lying supine for 20 minutes;
b). Actively changing postural position for 15 minutes;
c). Performing the hand-grip-test while seated, applying 30% of maximum voluntary-pressure for 3 minutes;
d). Performing the standard Valsalva´s maneuver for 20 seconds; and, finally,
e). Breathing deeply (six breaths per minute). Blood pressure was measured with a previously calibrated sphygmomanometer at the beginning and end of the rest period and in each of the other phases of the test.

   Power spectral analysis of the data was done in the manner described elsewhere (99,100) for R-R intervals recorded during the resting period, with the subject standing upright, and during normal pressure. This procedure yielded two main components, namely, low and high frequency (0,03 to 0,15 Hz and 0,17 to 0,42 Hz, respectively).

   The Valsalva´s maneuver results were evaluated by calculating the ratio between the longest R-R interval in phase 4 and the shortest in phase 2. The heart rate response to the deep breathing test was assessed as the mean difference between maximum and minimum heart rate during three successive respirations (101).

   For the statistical analysis, Student´s t test for paired comparisons, analysis of variance, the Scheffé test, and a multiple linear regression model with discriminant function analysis were used.
The results were as follows: (93,94)
A). The low-frequency spectral component of R-R interval variability, considered to be a primary and sensitive marker of sympathetic activity, increased in the active posture change test among the subjects in the control group (+ 30), but not in the chagasic patients (ChI,- 1 ±8, and ChII, - 2 ± 8 (P <0,05) (figure 10).

Figure 10. Power spectral analysis. Example of R-R variability in a normal subject and a ChII chagasic patients at rest and after standing. The increase in the low spectral frequency component (sympathetic) is seen only in normal subjects, this response being totally absent in chagasic patients (adapted form reference 94).


B). In the hand-grip test, which is regarded as an assessment of only sympathetic function, the low-frequency spectral component was significantly increased in the control group but not in the ChI and ChII patients (P <0,01) (Figure 11).
C). In Valsalva's maneuver the long R-R/short R-R ratio was significantly lower in the chagasic patients than in the control (P <0,05) (figure 11).
D). In the deep breathing test the ChII patients had a significantly lower ratio than the control group (P <0,001).

   On the basis of these results it was concluded that chronic chagasic patients unquestionably have alterations in both branches of the ANS -the sympathetic (evaluated by basal heart rate, the tilting test, and the hand-grip test) and the parasympathetic (assessed by Valsalva´s maneuver and the deep breathing test). These alterations do not derive from CHF, since they were present in patients (the ChI and ChII groups) who did not have the condition. The absence of a sympathetic response in the tilting test is identical to the results obtained when normal subjects are given ▀-blocking drugs (102).

Figure 11. Power spectral analysis. Example of R-R variability in a normal subject and a ChII chagasic patient at rest an during the hand-grip test. The low spectral frequency (sympathetic) component, which appears during the test in normal subjects but not in chagasic patients, can be clearly seen (adapted from reference 94).

ANTI-T. CRUZI AND ANTI-SCIATIC NERVE ANTIBODIES IN CHRONIC CHAGASIC CARDIONEUROPATHY
   It has been demonstrated that the immune response to T. cruzi plays a very important role in both the pathogenesis of Chagas' disease and protection against it (103-104). Antiheart (80, 105, 106) and antinerve antibodies have been reported in the serum of chronic chagasic patients (107-108).

  The sera of chronic chagasic patients contain antibodies that are highly reactive against T. Cruzi acid antigenic fractions, including a cytosolic fraction known as FIV (80). The Cruzipain antigen, a T. Cruzi cysteine-proteinase (109), cross-reacts with anti-FIV antibodies (110). The anti-FIV antibodies, in turn, are capable of recognizing in human cardiac tissue (80).

   In order to determine whether T. Cruzi infection could generate a humoral immune response against the FIV acid antigenic fraction, and to see whether that response could induce an autoimmune reaction against peripheral nervous components such as the constituents of the sciatic nerve, enzyme-linked immunosorbent assays (ELISA) and blotting were performed on 10 subjects in the control group and 36 chronic chagasic patients - 12 each in groups ChI, ChII and ChIII (110).

   The highest frequency of positive reactions against FIV was observed in the ChII patients, 92% of whom had positive titers (>400). In contrast, only 50% of those in groups ChI and ChIII were positive.

   Investigation of the autoimmune response to a saline extract of human sciatic nerve (a postmortem specimen from an individual with negative serology and blood type O Rh-) showed that the proportion of IgG binding to the nerve extract increased with the severity of the disease : ChI, 58%; ChII, 66% and ChIII, 75%. Treatment of the nerve extract with sodium periodate reduced the capacity of the antigens to bind IgG, thus demonstrating the hydrocarbonic nature of the epitopes involved.

   In studies to determine whether FIV and the sciatic nerve extract had cross-reacting epitopes, it was observed that the absorption of positive sera with FIV inhibited 48% to 69% of their reactivity against the nerve extract antigen. On the other hand, the absorption of positive sera with nerve extract inhibited only 12% to 23% of their reactivity against the FIV antigen.

   It was concluded that the ChII group was the group with "activity" - i.e., the one undergoing lesion "activity" - since the ChIII group, in which terminal fibrotic lesions had already been produced, had lower antibody activity (110). The FIV antigen and the peripheral (sciatic) nerve components shared some common epitopes. These epitopes were known to be basically carbohydrates, given their behavior in the presence of sodium periodate, which was similar to that described in other neuropathies (111). Hence, in Chagas' disease, autoreactivity against nerve components might be induced in infected subjects.

CLINICAL-IMMUNOLOGIC CORRELATION BASED ON ANTIGANGLIOSIDE ANTIBODIES IN CHRONIC CHAGASIC CARDIONEUROPATHY
   Gangliosides have generated great interest among physiologists and neurochemists, given their special physiochemical properties, the high concentrations that are found in the central and peripheral nervous system, and the selective alterations that they undergo in various neurological diseases (111-115). They have attracted the attention of those working on chronic chagasic cardioneuromyopathy (116) for the following reasons:
A). The infective form of T. Cruzi, the trypomastigote, is capable of modifying the surface chemical structure of epithelial cells in the myocardium and blood vessels by destroying sialic acid through the action of the enzyme neuraminidase (117).
B). Neuraminidase activity is elevated 10- to 20-fold when the parasite changes from (tyrpomastigote) (118). The coincidence of increased neuraminidase activity and infectivity in the trypomastigotes suggests that this enzyme is a factor in the virulence of the Chagas' disease agent (116, 118).
C). Gangliosides are found in high concentrations in plasma membrane and play a very important role in the membrane's function (119).
D). It has been established that ganglioside concentrations are three times higher in the cells of the heart's conduction system (which is frequently impaired in chronic chagasic patients) than in other myocardial cell membranes, and that the qualitative makeup of these gangliosides is different.
E). Neuraminidase cuts off the sialic acid residues of glycoproteins, oligosaccharides, and glycolipids (118). This may explain the role played by gangliosides in the nervous conduction system and terminal organs of chagasic patients (116, 120).

   For all these reasons, a comparative study was undertaken of antiganglioside antibodies in the three groups of chagasic patients with different degrees of cardiac compromise in order to determine whether there was a correlation between the frequency or levels of antiganglioside antibodies and the degree of clinical alteration.

   The study population consisted of 34 chagasic patients from groups ChI, ChII, and ChIII, ranging in age from 20 to 40 years. The group of 10 controls ranged in age from 25 to 40 years.

   The following purified gangliosides were used as antigen to detect the antibodies: GM1, GD1a, GD1b, GT1b (Fibia Research Laboratory, Abano Terme, Italy).

   The sera were tested using standard ELISA and dot blots (immune stains) methods. The samples were considered positive if the mean absorption level was more than three standard deviations higher than the mean value obtained in the parallel control sera.

The percentages of sera with IgG reactive to the gangliosides by ELISA are shown below: (Table 3)

   Antiganglioside IgM antibodies were present only against GT1b in one patient from the ChI group and another from the ChII group. Reactivity against gangliosides revealed by ELISA was confirmed by dot blots in all the patients. Only one patient who was negative against GM1 and GD1b by ELISA was positive by dot blots.

   Crossed-reactions between antitrypanosome and antigangliosideantibodies were also studied. absorption with GM1 eliminated 45% of the reactivity seen in nonabsorbed serum; absorption with T. Cruzi, 46%; and absorption with blood group B+ red cells, only 30%. These results suggest the following conclusions in regard to chronic chagasic cardioneuromyopathy :
a). There is a significant increase in the level and frequency of antiganglioside antibodies as the degree of cardiopathy progresses in chronic chagasic patients.
b). There is cross-reactivity between GM1 and T. Cruzi epimastigotes.
c). From these data it was not possible to determine whether the antiganglioside antibodies were the cause of the neuropathy, the consequence of the neurological lesion induced by the parasitic infection, or the result of a cross-reaction with T. Cruzi. What is clear is that the gangliosides play some role in chagasic cardioneuromyopathy (120-148).

GRATEFULNESS
To my teacher, the Dr. Hugo Palmero (+), to the Dr. Gabriel Schmunis for their support and to Mr. Mario Oscar Juárez (e-mail mario_o_Juarez@yahoo.com.ar) for the transcription of the present work.

*Contributions from a Workshop sponsored by the Pan American Health Organization / World Health Organization cosponsored by Association Internationale Pour la Recherche et I´Enseignement en Neurosciences / WHO Collaborating Center in Neurosciences / and Programa Nacional de Neurociencias / Consejo Nacional de Ciencia y Tecnología / Secretaría de Estado de Ciencia y Tecnología Argentina with the collaboration of Facultad de Medicina. Universidad de Buenos Aires and Universidad del Salvador, Buenos Aires, Argentina.

Scientific Publication n° 547 (Chapter 6, pages 99-148). Dr. Daniel Iosa M.D., Ph. D. PAN AMERICAN HEALTH ORGANIZATION. Pan American Sanitary Bureau, Regional Office of the WORLD HEALTH ORGANIZATION.

This modified contribution to the Second Virtual Congress of Cardiology (S.C.V.C.) has been authorized by the W.H.O. and P.A.H.O. (Dr. Gabriel Schmunis)

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2nd Virtual Congress of Cardiology

Dr. Florencio Garófalo
Steering Committee
President
Dr. Raúl Bretal
Scientific Committee
President
Dr. Armando Pacher
Technical Committee - CETIFAC
President
fgaro@fac.org.ar
fgaro@satlink.com
rbretal@fac.org.ar
rbretal@netverk.com.ar
apacher@fac.org.ar
apacher@satlink.com

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This company contributed to the Congress