Basic research models for the study of underlying mechanisms of electrical neuromodulation and ischemic heart-brain interactions

PROJECTS 2 AND 3: CARDIAC NOCICEPTOR ACTIVATION

The cortical structures and their related output pathways also serve as effector systems for initiation of autonomic and behavioral responses by forebrain neuronal networks that make us aware of cardiac pain. However, these cortical and subcortical structures involved in cardiac pain perception were more or less terra incognita. In addition, we studied fundamental aspects of cardiac nociceptor activation (Project 2) and transduction of cardiac pain (Project 3). Unfortunately, there was no experimental animal model for angina pectoris. The aim of these projects was to obtain, both in patients and in animals, knowledge about cardiac nociceptor activation mechanisms, the transmission and perception of cardiac pain, and behavioral and autonomic responses.

To enable the study of mechanisms of neurostimulation during episodes of acute cardiac pain, we worked out an animal model for angina pectoris. For that reason we experimented with models in which we created an acute myocardial infarction. We had to reject this model since surgery and, more importantly, anesthesia interfered with the patterns of cerebral expression of immediate early genes (c-fos, c-jun) triggered by cardiac pain and/or neurostimulation. However, a spinoff from this project was the observation that cardiac tissue damage causes a reproducible and selective cerebral endothelial leakage of immunoglobulin G (IgG) molecules. Follow-up experiments showed that proinflammatory cytokines, which are released into the circulation after cardiac tissue damage, can generate the same pattern of blood-brain barrier dysfunction⁷ (see Project 4).

We then experimented with infusions of capsaicin into the pericardial space of unrestrained and unanesthetized rats to induce acute cardiac pain. This model appeared to be very promising and allows visualization of the behavioral and autonomic responses to cardiac pain. Cerebral c-fos expression patterns, a marker for structures involved in cardiac pain transmission and perception, were studied and validated with positron emission tomography (PET) imaging in patients.⁸

Project 2: Nociception of cardiac pain in patients

To study relationships between neurotransmitters and other molecules that contribute to pain and psychological variables, we studied cardiac tissues obtained from 22 patients with angina during coronary artery bypass graft surgery (CABG). Cardiac nociceptor activation mechanisms were investigated in heart biopsies from these 22 CABG patients; reverse transcriptase polymerase chain reaction analysis (RT-PCR) was conducted for adenosine and bradykinin receptor mRNA.^9,10

An age-related decrease was observed in the adenosine A1 mRNA density but not in the bradykinin receptor mRNA levels. The adenosine A1 receptor density also correlated with pain characteristics reported in a questionnaire. Making use of semiquantitative RT-PCR, cardiac tissue substrates were assessed to determine the expression of adenosine A1 and bradykinin B1/2 receptor mRNA densities. The outcomes were associated with the quality of pain, age, gender, medication, and duration of disease.^9,10

For evaluation of pain characteristics, we used questionnaires and objective pain scores. We found that qualitative age-related alterations in angina perception correlated with the development of the more “strangling” component of angina at older age. This observation may be explained, in part, by a reduction in adenosine A1 receptor mRNA expression in the heart, since bradykinin B1/2 receptor densities remain the same.^9,10

Project 3: Nociception of cardiac pain in unrestrained rats

Having identified neural pathways, we studied neurons that were activated during electrical neuromodulation. ¹¹ In search of a putative mechanism of action of electrical neuromodulation, we hypothesized that neuromodulation affects processing of nociceptive information within the central nervous system (CNS). To characterize neural activity we used expression of both the immediate early gene c-fos and the “late gene” or stress protein known as heat shock protein 72 (HSP72). c-fos was used to identify structures in the CNS affected by spinal cord stimulation. HSP72 was applied to ascertain whether spinal cord stimulation might operate as a stressor.¹²

Animal experiments were conducted on unrestrained unanesthetized rats implanted with a permanent catheter in the pericardial space; acute cardiac pain was triggered in this space using capsaicin as the algogenic substance.¹³ The autonomic cardiovascular responses were recorded with implantable telemetric devices. Behavioral responses were recorded on videotapes taken from the same animals in which the involved cerebral structures were characterized by analyzing cerebral immediate early gene expression. Quantification of data makes it possible to study the effects of electrical neuromodulation and analgesic drugs on perception of cardiac pain. To apply electrical neuromodulation, two electrodes were positioned and sutured epidurally at the spinal cord of the rats. One electrode was fixated at spinal nerve C7 and the other at T2. Furthermore, we studied the effect of spinal cord stimulation on behavior. Three hours after stimulation, the rats were sacrificed and their brains and spinal cords were removed.

The treated group showed regional increased c-fos expression in a select group of regions of the limbic system—periaqueductal gray, paraventricular hypothalamic nucleus, paraventricular thalamic nucleus, central amygdala, agranular and dysgranular insular cortex, (peri)ambiguus, nucleus tractus solitarius, and spinal cord—involved in the processing of pain and cardiovascular regulation, among other functions. Moreover, in both treated rats and controls, HSP72 expression was found in the endothelium of the enthorhinal cortex, the amygdala, and the ventral hypothalamus, but not in the neurons. The treated animals were significantly more alert and active than were the controls.

Thus, the rat model we developed appears to be suitable for studying potential mechanisms through which neuromodulation may act. Moreover, neuromodulation affects c-fos expression in specific parts of the brain known to be involved in regulation of pain and emotions. HSP72 expression is limited to the endothelium of certain parts of the CNS, and thus physical stress effects were excluded as a potential mechanism of neuromodulation. Finally, our experimental model identified regions corresponding with regional cerebral blood flow changes during neurostimulation in patients.⁸