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Am J Physiol Regul Integr Comp Physiol 287: R1468-R1477, 2004. First published August 5, 2004; doi:10.1152/ajpregu.00251.2004
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Left vagal stimulation induces dynorphin release and suppresses substance P release from the rat thoracic spinal cord during cardiac ischemia

Fang Hua,1 Jeffrey L. Ardell,2 and Carole A. Williams1

Departments of 1Physiology and 2Pharmacology, College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614

Submitted 15 April 2004 ; accepted in final form 2 August 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Electrostimulatory forms of therapy can reduce angina that arises from activation of cardiac nociceptive afferent fibers during transient ischemia. This study sought to determine the effects of electrical stimulation of left thoracic vagal afferents (C8–T1 level) on the release of putative nociceptive [substance P (SP)] and analgesic [dynorphin (Dyn)] peptides in the dorsal horn at the T4 spinal level during coronary artery occlusion in urethane-anesthetized Sprague-Dawley rats. Release of Dyn and SP was measured by using antibody-coated microprobes. While Dyn and SP had a basal release, occlusion of the left anterior descending coronary artery only affected SP release, causing an increase from lamina I-VII. Left vagal stimulation increased Dyn release, inhibited basal SP release, and blunted the coronary artery occlusion-induced release of SP. Dyn release reflected activation of descending pathways in the thoracic spinal cord, because vagal afferent stimulation still increased the release of Dyn after bilateral dorsal rhizotomy of T2–T5. These results indicate that electrostimulatory therapy, using vagal afferent excitation, may induce analgesia, in part, via inhibition of the release of SP in the spinal cord, possibly through a Dyn-mediated neuronal interaction.

antibody-coated microprobes; angina; cardiac nervous system; analgesic peptides; nociceptive peptides

INTRACTABLE ANGINA CAN BE one of the more debilitating forms of pain experienced. While myocardial ischemia involves both local changes in myocyte function and reflex alterations in the cardiac nervous system that regulates the myocardium, the perception of angina involves the excessive activation of cardiac sympathetic afferent neurons (16, 35). These multimodal sensory afferent neurons respond to ischemia-induced changes in the chemical/mechanical milieu of the heart (16, 35). The cell bodies of these afferents are found in the dorsal root ganglia of spinal segments C8–T9 (cervical 8-thoracic 9), with the majority associated with spinal segments T2–T6 (29) and project mainly to laminae I, V, VII, and X (29). Activated cardiac sympathetic afferent fibers excite cells in the spinal thalamic tract (STT) primarily in T1–T6 spinal segments (4, 41) and C1–C2 segments (8), as well as other ascending tracts, including the spinoreticular, spinomesencephalic, and spinosolitary tracts (16).

Clinical treatment for ischemic heart disease focuses on relieving the angina through a hierarchy of treatment modalities, starting with lifestyle changes and progressing through pharmacologically induced coronary vasodilation, angioplasty, and, finally, coronary bypass procedures. Some individuals, however, are refractory to these procedures and have become candidates for emerging, alternative neuromodulation therapy (32). Neuromodulation therapies include both transcutaneous and direct nerve electrical stimulation (32), including low-intensity electrical stimulation of left vagal afferent fibers at the cervical-thoracic level (1, 42–44, 54, 55) and spinal cord stimulation (15, 16, 34). While electroneuromodulation therapy is not currently widely used clinically in this country for angina, the electrophysiological effects of these approaches have been documented in a number of animal studies (1, 7, 16, 34, 42–44). Moreover, clinical experience in Europe indicates that such therapies are effective treatment options for patients with severe angina who are refractory to conventional antianginal therapy and who are not candidates for revascularization therapy (15, 32).

Insights into the basic mechanisms of left vagal stimulation (LVS) as a treatment modality for angina derive primarily from electrophysiological studies describing changes in the activity of STT neurons to noxious stimuli (7, 44). A number of studies have looked at the changes in electrical activity of cells in the STT that were excited by noxious stimuli and subsequently modulated using stimulation of cervical-thoracic vagal fibers. Ren et al. (44) report that the spontaneous activity of 36% of STT neurons was inhibited by all intensities of vagal stimulation. Chandler et al. (7) indicate that thoracic vagal stimulation inhibited STT neurons in spinal segments below C3, but excited 46% of the cells in C1–C3. Furthermore, vagal afferent stimulation was found to decrease the cardiac-evoked total motor unit potentials in anesthetized rats (27). Similar electrophysiological changes are reported in response to spinal cord stimulation (16, 34).

While much is known about the specific cells and pathways excited by cardiac ischemic-sensitive nociceptive afferent neurons, little information exists regarding specific neuromediator(s) of the nociceptive signal at the spinal level. Recently, we reported that activation of cardiac ischemic afferent neurons by occlusion of the left anterior descending artery induced the release of substance P (SP) from a number of laminae in the thoracic spinal cord (at the T4 level) (23). This led us to propose that heterogeneous activation of cardiac afferents, as occurs during coronary artery occlusion (CoAO), represents an optimum signal for stimulating neuronal pathways that lead to the perception of angina (23). Furthermore, we proposed that SP serves as a primary nociceptive peptide at the spinal level (23). This suggestion is supported by the following: first, SP coexists with glutamate in primary afferent C fibers (9); second, SP is found in high concentrations in the spinal cord and is released during stimulation of visceral and somatic C-fiber afferent nerves (11, 13, 14, 30); third, spinal tachykinin receptors are activated during nociceptive signaling (10); and fourth, myocardial ischemia activates dorsal root ganglia neurons that are also stimulated by SP (9, 24).

Stimulation of left thoracic vagal fibers decreases the activity of many of the cells in the same dorsal horn sites that are excited by activation of cardiac ischemic-sensitive afferent neurons (7, 44). Because we showed that SP was released in the thoracic spinal cord in response to cardiac ischemia (23), it was of interest to determine, first, whether activation of cardiac ischemic-sensitive afferent neurons also induced the release of an analgesic (i.e., opiate) peptide in the thoracic spinal cord; second, whether electrostimulatory neuromodulation suppressed the cardiac ischemic-sensitive afferent fiber-mediated release of SP in the thoracic spinal cord; and, third, whether this stimulation induced the release of an analgesic peptide, such as an opiate.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Surgical preparation of animals. Sprague-Dawley rats (n = 27) of either sex (average weight: 328 ± 7 g) (Harlan, Indianapolis, IN) were anesthetized with urethane (1.5 g/kg body wt ip). Surgical level of anesthesia was maintained via supplemental injections of urethane (30 mg/kg) through the right jugular vein hourly, or more frequently as needed, during the experiment. All procedures and experimental protocols were reviewed and approved by the Institutional Committee on Animal Care and Use and conformed to the Animal Welfare Act, according to the Public Health Policy on Humane Care and Use of Laboratory Animals. Body temperature was maintained at 37°C by placing the animal on a heating pad. The left femoral artery was cannulated for arterial pressure measurement, and a tracheotomy was performed so that the rats could be ventilated with room air using a small-animal ventilator (Harvard Instruments) at frequencies of 70–80 breaths/min and a tidal volume of 2–3 ml.

The mean arterial pressure (MAP) was calculated from the diastolic pressure plus one-third the pulse pressure. Heart rate (HR) was either determined from the pressure pulse signal and displayed on a Grass chart recorder via a tachograph or calculated from the ECG recording. Data presented in Table 1 are means ± SE. For hemodynamic data, both the initial response to the experimental intervention as well as the steady-state levels (taken 1 min after initiation of the intervention) are reported in Table 1. Only one experimental intervention (i.e., coronary occlusion or vagal stimulation, etc.) was performed on a given group of rats, with each intervention repeated twice for a total of three interventions for each animal. The group mean reported in Table 1 was derived from the average response of the repeated intervention for each animal. There was no difference in the blood pressure (BP) or HR responses for the first compared with the third intervention in any given animal. Thus the mean BP and HR levels were averaged for each rat, and this value was entered into the group mean. Significance was determined by using (SigmaStat) Student's t-test for paired data within a specific experimental group (for rest vs. intervention or intervention vs. recovery) and by using one-way ANOVA for between-group analysis with subsequent follow-up comparisons using Tukey's test. P ≤ 0.05 is taken as the minimum level of significance.


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  		Table 1. Cardiovascular responses to vagal afferent fiber stimulation

 
Laminectomy. The spinal cord from T1 to T6 was exposed on all of the rats used in these experiments by removing the corresponding vertebral processes. This was done to allow placement of the antibody-coated microprobes. An intravenous injection of turbocurarine (67 µg/kg body wt) was given before removal of the dura and pia maters from these segments. The exposed spinal cord was kept moist and warm by superfusing sterile, oxygenated, artificial cerebrospinal fluid (in mM: 125 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 3.7 dextrose; 6 urea, 0.1% BSA) over the area, wrapping the area with a piece of plastic film, and positioning a heat lamp over the area. The animal was fixed in a Kopf stereotaxic frame, and the spinal cord was maintained in a fixed position by the use of spinal clamps secured to the vertical processes of segments both rostral and caudal to the laminectomy. Some of the rats (n = 7) also underwent a dorsal rhizotomy (dR). The purpose of the dR was to determine whether afferent input from the ischemic myocardium altered the release of an analgesic peptide, because we found that thoracic rhizotomy significantly attenuated the release of SP (see Ref. 23). In these cases, the lateral processes of T1–T6 were completely removed. The dorsal roots of spinal segments T2–T5 were identified, gently separated, and then sectioned bilaterally, close to the lateral most area of each segment. A 60-min rest period was allowed following completion of all surgical procedures before the experimental protocols involving either CoAO or LVS were initiated (see below). For animals with dR, the LVS protocol was followed as described below for animals with intact dorsal roots (see LVS).

CoAO. A left thoracotomy was performed between the fourth rib following initiation of ventilation (n = 7 rats). A segment of saline-soaked 5–0 suture was looped around the left anterior descending coronary artery, near its branch point from the left coronary artery. The ends of the suture were passed through a 1-in. length of double-barreled polyethylene tubing. The ends of the tubing were rounded so that no rough surface of the tubing would damage the coronary artery. The tubing was gently placed next to the heart and secured by three knots tied on the suture at the external end of the tubing length, with the last knot up against the end of the tubing. The tubing and suture assembly was then externalized, and the thorax was closed. CoAO was accomplished by advancing the last knot up against the external end of the tubing, 2 mm away from the tubing. This permitted reproducible occlusion of the left anterior descending artery without tearing the vessel. Either a dynorphin (Dyn) or SP antibody-coated microprobe was positioned in the T4 spinal level (see below) for 10 min ("rest" probe) followed by a second and then third rest probe inserted for subsequent 10-min periods before the beginning of the occlusion sequence. CoAO was applied sequentially for 90 s with a 60-s rest interval over a 10-min period (i.e., four occlusions were applied over the 10-min period). This constituted the coronary occlusion intervention, and a single microprobe remained in the spinal cord during this 10-min period. A "recovery" probe was placed in the spinal cord at the completion of the occlusion intervention for 10 min. After a 30-min rest, the entire coronary occlusion procedure was repeated twice in each animal (with a fresh "coronary occlusion" probe and a fresh recovery probe used for each period).

LVS. Vagal nerves were isolated at the level of the C8–T1 spinal segments and sectioned bilaterally (n = 7 rats). A platinum bipolar electrode was placed on the central end of the sectioned left vagus and fixed with Kwik-Cast (World Precision Instruments). The left vagus was used because it has more afferent fibers than the right (22). LVS was applied by using a Grass constant-current stimulator with an isolation unit using 25 V, 0.2 ms at 10 Hz (1). Stimuli were delivered sequentially for 90 s with 60-s rest intervals over a 10-min period (i.e., 4 stimuli trains). The LVS procedure was repeated twice in each animal. Separate probes were placed in the thoracic spinal cord for 10 min each during rest, before the electrical stimulation, for 10 min during the stimulation procedure, and for the 10-min recovery period, immediately after the stimulation procedure, as described above for the CoAO protocol. For animals with bilateral dR (T2–T5), a similar LVS protocol was followed.

Combined CoAO and LVS. The left anterior descending coronary artery and vagi were prepared as outlined above (n = 6 rats). Concurrent left anterior descending coronary occlusion and LVS were applied for successive 90-s intervals with 60-s rest periods over a 10-min period and repeated twice in each animal. Microprobes were placed in the thoracic spinal cord, as described for the other protocols.

Measurement of immunoreactive SP and Dyn using immobilized antibody microprobe technique. The release of endogenous immunoreactive SP (irSP) and immunoreactive Dyn (irDyn) from sites in the thoracic spinal cord was measured by using the antibody-coated microprobe technique, as previously described (12, 50). Each probe was inspected to verify that the shafts were straight. Glass microelectrodes (tip diameter ~10 µm; shaft diameter 2 mm from the tip, ~20–30 µm) were coated as described before and incubated for 24 h at 4°C with protein A (Sigma Chemical) before the experiments (50). Probes were then incubated with their respective antibodies [for either SP or Dyn A-(1–13)] for two 24-h periods at 4°C in 5 µl of a 1:1,000 dilution (Phoenix Pharmaceuticals) in PBS-azide solution, pH 7.4, with the solution changed after the first 24 h. The SP antibody was determined not to react with neurokinin A, B, or K. The Dyn antibody had negligible cross reactivity (≤1.5%) with Dyn-(1–10) or -(1–11). We also tested whether endomorphin-2 was released from the thoracic spinal cord. Antibody to endomorphin-2 (Phoenix Pharmaceuticals, 1:1,000) was incubated with microprobes, as described for SP and Dyn (cross reactivity with endomorphin-l, <10%). In all experiments where probes were inserted into the thoracic spinal cord, a set of control probes (designated as in vitro probes) was identically and simultaneously prepared as the in vivo probes. These in vitro probes were used to determine the sensitivity of the binding of radiolabeled ligand (125I-Tyr8 SP or Dyn, Phoenix Pharmaceuticals) (see Ref. 12 or 50) and to confirm the uniformity of binding of the silane and antibody along the shaft of the probes. Typically, 10–7 M SP will displace ~50% of the labeled SP, and 5 x 10–7 M Dyn will displace ~50% of the labeled Dyn from approximately a 1-cm length of a probe tip. Dose-response trials indicate that, between 10–9 M and 10–10 M, unlabeled ligand will give statistically different counts than buffer alone when incubated with probes. Each in vivo experiment utilizes probes from different batches, so any differences in coating are randomized, and the results from no one category or group of probes (e.g., rest or "stimulus," etc.) would be skewed.

Each of the in vivo probes was positioned at the midpoint of the T4 spinal segment (in terms of its length) at 0.5 mm lateral to the midline and inserted into the cord to a depth of 2 mm below the dorsal surface. Each probe remained in situ for 10 min, for the duration of either the rest, the experimental procedure (coronary occlusion and/or LVS, as described above), or the recovery periods. New probes were used for each 10-min pre- or postexperimental procedure and each experimental procedure, and these were designated as rest, coronary occlusion (CoAO), LVS, coronary occlusion with LVS (LVS+ CoAO), or recovery. A total of 247 probes were used and analyzed for these experiments. Each probe was calibrated against the midline and surface of the spinal cord, and placement of each probe was performed by using a stereotaxic surgical microscope to ensure repeatability of their placement. At the completion of the experiment, 9.7–13.4 nl of Pontamine blue dye were deposited through the last probe via an automated positive-pressure injection system (Nanoject II) to verify position of the probe tips. This was done by visualizing the dye in 30-µm cross sections of the spinal cord at the completion of the experiments, using a stereotaxic atlas (39). The deposit of blue dye was visualized through an Olympus OM-3 microscope, and the image of the spinal section was captured through the image analysis setup following distance calibration. The image was then compared with the standardized section in the reference atlas (39).

At the completion of the 10-min in situ time, probes were withdrawn from the spinal cord, washed briefly in ice-cold PBS, and incubated with radiolabeled SP or Dyn (0.01 µCi/5 µl PBS, 7.4) for 24 h at 4°C. Probes were washed in ice-cold PBS-Tween (0.05% Tween 20; Fisher) for 15 min under vacuum manifold to remove any radioactive solution that may have been on the inside of the probe. A 1-cm length of the probe, starting from its tip, was broken off, secured to a piece of heavy paper, and counted for radioactive binding. The shaft ends of the tips were secured to a sheet of paper and placed against monoemulsion X-ray film (Kodak, Biomax MR-1) for 3 days. The set of in vitro probes, incubated with the same amount of radiolabeled ligand, was washed in the Tween-PBS and exposed with the same sheet of X-ray film as the in vivo probes.

Image analysis of the microprobes. Autoradiographic images of the microprobes were analyzed for patterns of inhibition of binding of the radiolabeled ligand along the length of the probe. Such inhibition is indicative of where unlabeled (e.g., endogenously released) SP or Dyn bound to the antibodies on the probe during the in vivo exposure time. Because the position of the probe tips was marked by a deposit of blue dye in the spinal cord, sites of release of the peptide could be determined from differences in the optical densities of the probe images on the X-ray film (see Fig. 4). The analysis was carried out based on initial methods described by Hendry et al. (21) and modified by this laboratory. A computerized image analysis system (MCID, Imaging Research) was used to linearly integrate the images transversely in 16-µm increments for the total 4-mm length of the probe, starting from the probe tip. Background grayness, due to the exposed X-ray film alone, was subtracted from each pixel of the probe image.



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  		Fig. 4. Sites of release of Dyn. Projection drawing of T4 spinal segment showing boundaries of dorsal horn laminae superimposed through projection drawings using stereotaxic atlas (39) as reference. Student's t-test from Fig. 3A, showing the difference in image analysis graphs between the rest and LVS probes, is superimposed on the section. The t-test is displaced to the right of the midline for illustrative purposes so that sites within the dorsal horn where the differences between these two sets of probes occurred could be identified. Areas showing release include the superficial laminae I-III, V, and VII. Pseudocolor image of Dyn antibody-coated microprobe during LVS is superimposed to the right of the section for illustrative purposes as well, and its location in the diagram is not representative of location of probes inserted into the spinal cord to capture peptide release. Note the lower intensity binding of radiolabeled Dyn, corresponding to the area along the length of the probe where there were significant differences in the gray levels between the rest and LVS probes. Average location of probe tips within the T4 segment is represented by the solid dot symbol (vertical and horizontal bars = SE; n = 27). Dashed line indicates position of probes within the spinal segment. Scale bar = 0.5 mm (applies to spinal segment, probe image, and t-test).

 
In the diagrams presented, the mean optical density of the probe image is converted to a gray scale in arbitrary units of 0–1,026 (with 1,026 being the darkest gray level). Each probe image was analyzed for 4 mm: the first 2 mm, starting at the tip, corresponded to the segment of the probe inserted into the spinal cord (designated 2 to 0), whereas the next 2 mm corresponded to the part of the probe that remained outside the spinal cord (designated as 0 to –2 mm). The 2 mm outside the spinal cord served as an internal control area along each probe and for between-group controls via comparison to similar segments on the in vivo and in vitro probes. The data presented in the image analysis of Figs. 1–5 are given as the mean gray levels ± SE for each specified group of probes. Differences in the patterns of binding of radiolabeled SP or Dyn A-(1–13) along the probes during various experimental interventions were determined by Student's t-test for paired data. The calculated t-value, where P = 0.05 (the minimum level of significance taken), is plotted along the lower portion of the image analysis graphs (just above the abscissa). The t-value for each pixel along the analyzed image was calculated and plotted in relation to the t-value of P = 0.05. Any points along the length of the probes that were different from each other appear above the t-value line and indicate significance. Because the resolution of detecting a difference in the binding of radiolabeled peptide is of the order of 100 µm (12), biological significance was defined only when the difference between two groups (i.e., the t-value) was maintained above the P = 0.05 line for a linear distance of at least 100 µm. Differences that appeared above the t-value line but were <50–100 µm in length were not considered to be a biological event. This technique determined whether either SP or Dyn was released, what specific sites in the spinal cord released these neuropeptides, and whether an experimental intervention altered the spatial pattern of release of these peptides within the T4 segment of the spinal cord. This was possible because the tips of the probes were located by identification of the deposited blue dye in the histological sections of the spinal cord using a reference atlas (39). This technique is not used to quantify differences in the amounts of peptides released, and this should not be inferred from the data presented (e.g., the t-value above the significance line).



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  		Fig. 1. Pseudocolor microprobe images. Images are of dynorphin (Dyn) antibody-coated microprobes as they appeared from exposed X-ray film. Pseudocolor was automatically applied to the gray levels of autoradiographic probe images converted from optical densities. All probes are viewed with tip ends facing leftward. All in vivo probes were inserted into T4 spinal segment 0.5 mm lateral to the midline and to a depth of 2 mm. A total length of 4 mm was analyzed (full 4-mm image presented in each panel): 2 mm from the tip inside the spinal cord and the immediate 2 mm that remained outside the spinal cord. Corresponding regions were analyzed on matched in vitro probes. A: probe inserted into T4 at rest, before coronary occlusion. Note the difference in intensity of the image in the first 2 mm of the probe compared with the in vitro control probes (F). B: probe inserted during coronary artery occlusion (CoAO). C: probe inserted in T4 during left vagal stimulation (LVS). D: probe inserted during CoAO plus LVS. E: probe inserted during LVS following dorsal rhizotomy (dR) of spinal segments T2–T5. F: example of control in vitro probe not inserted into the spinal cord. Scale bar shown in F = 1 mm (applies to all panels). Arrowheads in A–E: to the left indicate part of the probe that was in the T4 spinal cord, and to the right indicate the part of the probe that remained outside the spinal cord. Note similarities of the outside segments (rightside 2 mm) of the in vivo and in vitro probes. Probe images in A, B, and F are from same experiment. Probe images in C, D, and E are from different rats and different experiments, as indicated in the panels.

 


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  		Fig. 5. Image analysis graphs of substance P (SP) microprobes. A: comparison of the rest probes inserted into T4 to in vitro probes. B: comparison of rest probes to probes inserted into the spinal cord during LVS. C: comparison of rest probes to probes inserted into T4 during simultaneous CoAO and vagal stimulation, CoAO + LVS. D: comparison of probes during CoAO to probes during CoAO + LVS.

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of CoAO on Dyn release. Insertion of microprobes into the spinal cord had no effect on BP or HR. Applying coronary occlusion caused a modest hypotension and tachycardia, as seen in Table 1. Sham-operated animals (for the CoAO procedure, n = 4) had resting MAP of 122 ± 9 mmHg and HR of 390 ± 21 beats/min. Although the resting MAP and HR in the CoAO group were lower than those in the sham group, these differences were not significant (MAP, P = 0.08, F = 3.97; HR, P = 0.70, F = 0.158), except for the animals undergoing LVS following rhizotomy (see Table 1). Typical autoradiographic images of Dyn antibody-coated microprobes are shown in Fig. 1. Compared with the in vitro control probe (Fig 1F), a probe inserted into the T4 spinal cord during rest periods shows a less dense image from its tip to ~1.5–2.0 mm from its tip (Fig. 1A), indicating inhibition in binding of radiolabeled Dyn along this part of the probe. The segment of the probe that remained outside the spinal cord (i.e., the next 2 mm) has a uniform intense image that resembles the appearance of a control in vitro probe, as seen in Fig. 1F. Insertion of microprobes during CoAO also revealed a less intense binding for the 2 mm of the probe that remained in situ for the 10 min of intermittent occlusion of the left anterior descending coronary artery (see Fig. 1B) compared with the control in vitro probe (Fig 1F).

Summary image analysis graphs showing the average gray levels of rest probes compared with in vitro probes are presented in Fig. 2A. The gray levels of the 2 mm of the rest probes that were in the spinal cord were less intense compared with the gray levels of the same length segment of the control in vitro probes (i.e., upward shift), indicating an inhibition in binding of radiolabeled Dyn, thus a basal release of endogenous irDyn from laminae I-VII in the dorsal horn. Figure 2B shows the gray levels of the rest probes, and the probes in the spinal cord during CoAO were not different from each other (i.e., no length of the 2 mm of the rest or CoAO probes had t-values greater than P = 0.05). This indicates that no additional irDyn was released in response to CoAO. Correspondingly, the gray levels of the rest and recovery probes were also virtually identical, indicating similar patterns of endogenous Dyn release were evident postcoronary occlusion, compared with baseline rest (see Fig. 2C).



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  		Fig. 2. Comparison of the binding patterns of radiolabeled Dyn to microprobes and the resulting differences in optical density [converted to gray level in arbitrary units (au), with 1,026 being the darkest] of autoradiographic probe images. Gray levels are given as the means ± SE (dotted traces above and below the main traces) and are plotted against the length of probe inserted into the spinal cord (2–0 mm) together with the 2-mm length that remained outside the spinal cord (0 to –2 mm). Plots of the t-value (solid blue line, P = 0.05 minimum level of significance) are superimposed just above the abscissa in each panel. Areas along the length of the probes where the difference in the mean gray levels were equal to or greater than P = 0.05 indicate significant differences between the corresponding two sets of probes. A: comparison of the rest probes, before CoAO, to the in vitro probes. B: comparison of rest probes and probes inserted into the spinal cord during CoAO. C: rest probes compared with recovery probes post-CoAO.

 
Effect of LVS on Dyn release. Applying electrical stimulation of the central end of the left vagus at the cervical-thoracic junction in this preparation, with bilateral vagotomy, lowered BP but had little effect on HR (see Table 1).

LVS increased the release of irDyn from the thoracic spinal cord above the resting levels. Note the diminished image intensity in Fig. 1C (i.e., the 2 mm in situ segment) compared with a typical rest probe (seen in Fig. 1A). Figure 3A summarizes the effect of LVS and indicates that the vagal stimulation-induced increased release of Dyn was evident from 0 mm (i.e., surface of spinal cord) to ~1.2 mm in the spinal cord. This corresponds to laminae I-V and the dorsal aspects of lamina VII as the sites of release of irDyn, as shown in Fig 4. Data from this set of experiments also indicated that the sites of irDyn release corresponded with neuronal soma that stained Fos positive in response to the LVS stress (22a). We also tested whether the opiate endomorphin-2 was released from the T4 spinal level in response to left vagal afferent stimulation. While there was a basal release of endomorphin-2 from laminae I-III, V, and VII (n = 12 rest probes, n = 12 in vitro probes), the LVS did not induce further detectable release of endomorphin-2 (n = 12 rest probes, n = 13 LVS probes; graph not shown).



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  		Fig. 3. Image analysis graphs of Dyn microprobes during LVS ± CoAO procedures. A: comparison of the binding patterns of rest probes to probes inserted into T4 during LVS. B: comparison of rest probes to probes in the spinal cord during simultaneous LVS + CoAO. C: comparison of probes during CoAO to probes during CoAO + LVS. D: comparison of rest probes following dR compared with probes during LVS after dR (LVS + dR). E: comparison of probes during LVS compared with probes during CoAO applied during LVS (CoAO + LVS). F: comparison of probes during LVS compared with probes during LVS following dR (LVS + dR). Details of graphs are as described in Fig. 2 legend.

 
Subsequent experiments evaluated the potential for interaction between LVS and CoAO for Dyn release. In a separate group of rats from those described in Fig. 3A, Fig. 3B shows that the differences in the gray levels between the rest probes compared with the probes during simultaneous CoAO and LVS occurred along the same segment length (i.e., 0 to ~1.3 mm) as the differences observed between rest and LVS probes (shown in Fig. 3A). This finding indicates that application of CoAO during LVS did not alter the sites of release of irDyn from T4 spinal segments. Furthermore, the differences in the gray levels at this same segment length (i.e., 0 to ~1.3 mm) are maintained when comparing the binding profiles of the CoAO probes to the CoAO + LVS probes (see Fig. 3C).

In the next group of rats, the LVS procedure was performed following bilateral dR of T2–T5 segments. Rhizotomy tended to result in a higher resting BP than the vagal stimulation group, but this difference was not significant (P = 0.73). While the mean steady-state BP in the vagal stimulation + dR group was higher than the LVS alone group, this was not significant (P = 0.29) (see Table 1). As seen in Fig. 3D, LVS still caused less binding of radiolabeled Dyn to probes in the thoracic spinal cord. The same differences between the rest probes and the corresponding in vitro probes (graph not shown) occurred for this group of rats as for the control condition shown in Fig. 2A (in rats with intact dorsal roots). These data indicate that dR did not affect the basal release of irDyn from the T4 spinal cord. LVS continued to induce Dyn release following rhizotomy (see Fig. 3D). This finding suggests that the LVS is the initiating event causing the release of endogenous irDyn in the thoracic spinal cord. This notion is supported by the data shown in Fig. 3E, which compares the binding patterns of the LVS probes during simultaneous CoAO and LVS. There was no difference between these two groups of probes, again indicting that cardiac ischemia, i.e., CoAO, was not the stimulus for increasing irDyn release from the thoracic spinal cord. Furthermore, the binding profiles of probes during LVS in rats with dR (see Fig. 3F) indicate that LVS induces irDyn release, albeit the release was shifted slightly to deeper lamina within the T4 spinal level compared with rats with intact dorsal root afferent input. Together, these data indicate the predominance of descending projections in mediating the spinal cord neuronal release of Dyn initiated by stimulating vagal afferent axons during LVS.

Effects of LVS on SP release. As observed previously (23), there is a background release of SP from the thoracic spinal cord (Fig 5A, comparing in vivo rest vs. in vitro probes). LVS suppressed this basal release of irSP from the thoracic spinal cord from laminae III-VII (Fig. 5B). Even though CoAO increased release of irSP from laminae I-VII (Fig. 5D), applying LVS concurrently with coronary occlusion blunted SP release in these regions (Fig. 5, C and D). In addition, the hemodynamic changes (hypotension and tachycardia) accompanying coronary occlusion were mitigated by preemptive LVS (Table 1).


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Refractory angina affects a considerable number of individuals. Those who suffer from this condition and have inadequate relief from the pain following classical surgical and/or pharmacological clinical treatment might be candidates for electroneuromodulation therapy (28, 32–34). Two such approaches that have been tried are vagal afferent or spinal cord stimulation (18, 28, 54–55). Both are reported to reduce intractable angina with no adverse cardiovascular consequences (18, 28, 32, 42, 43, 54, 55). Widespread clinical studies are limited, and how such approaches exert their antianginal effects is not well defined, but the mechanisms likely involve both cardiac (18, 28, 54, 55) and neuromodulator (18, 28, 34, 38, 55) components. While the electrical stimulation vs. coronary bypass study indicates that neuromodulation therapy is an effective treatment option for patients with chronic refractory angina (15), further and more extensive clinical investigation would be needed to verify the efficacy of these techniques as an antianginal therapy. Moreover, additional basic studies are required to determine the underlying targets (central vs. peripheral) that are modified by each specific type of neuromodulatory therapy. The systems controlling the cardiovascular responses to activation of cardiac ischemic-sensitive afferent neurons involve both central and peripheral autonomic and intrinsic-cardiac neural adjustments; i.e., there is a hierarchy of synergistic interactions at multilevels in the central nervous system (2). Electromodulatory interventions, such as left vagal afferent nerve stimulation, most likely impact the processing of these control mechanisms at the upper cervical spinal cord level (C1–C3) as well as the brain stem (18) and influence the responses to the input signals from the heart at the thoracic spinal (T2–T6) level (18).

There are a number of electrophysiological studies that show that cervical-thoracic vagal afferent stimulation inhibits STT neurons, not only in the thoracic spinal cord in response to cardiac ischemia (1, 8, 40), but also in the lumbar and sacral spinal levels in response to viscerosomatic noxious stimuli (1, 8, 16–19, 40). There is, however, virtually no information about the neurochemical mediation of visceral afferent nociceptive signaling or its modulation by electrostimulatory techniques. This study offers the following major findings: first, that activation of cardiac ischemic-sensitive afferent neurons by CoAO increases release of irSP from the thoracic spinal cord (at T4); second, that CoAO, in and of itself, does not increase detectable changes in the release of Dyn from T4; third, that left vagal afferent axonal stimulation inhibits the release of SP from the thoracic spinal cord; fourth, that left vagal afferent stimulation applied during CoAO blunts cardiac afferent-mediated release of SP from the thoracic spinal cord; and fifth, that LVS increases the release of Dyn from the thoracic spinal cord and this release is likely mediated via descending pathways into the thoracic spinal cord. The origin of this descending projection, whether from higher cervical spinal segments or supraspinal sites, remains to be determined. Taken together, these data indicate that the antianginal effect of electromodulation with vagal afferent stimulation is mediated, in part, within the spinal cord by a neuronal mechanisms involving the suppression of SP release by the endogenous opiate, Dyn A.

Differential activation of left ventricular nociceptive afferent neurons by transient focal ischemia increases the release of the putative neurotransmitter SP, which, in turn, may activate STT cells in the thoracic spinal cord (23). STT neurons project to upper cervical spinal segments as well as higher centers in the brain to process the pain associated with myocardial ischemia (16, 35). Cardiac ischemic-sensitive afferent neurons are primarily a subset of sympathetic afferent nerves that enter the spinal cord at the thoracic level, with a concentration at the T1–T4 spinal segments (16, 18). However, other cardiac ischemic-sensitive afferent neurons are vagal afferents that project to nucleus tractus solitarius (NTS) neurons that, in turn, influence neurons in the C1–C3 spinal segments (16). There are a number of descending inhibitory pathways that regulate the activity of neurons in the thoracic spinal cord (16, 35), including one that originates from the NTS (16, 35). One important pathway that may be relevant to the study conducted here involves excitation of upper cervical cells, via propriospinal circuits, that stimulate interneurons, which inhibit neurons in the thoracic (and lower) spinal segments (16, 18).

Previous results from this laboratory showed an extensive number of Fos-positive cells in the cervical as well as thoracic spinal cord in response to CoAO (22a). The majority of the cells activated were found in laminae I-VII and X, and more cells were activated in the medial portions of the dorsal horns than the lateral portions. In the present study, we also observed a basal, resting release of irSP from the dorsal horn sites in the thoracic spinal cord, and, as we reported previously, CoAO increased such release (23). dR of spinal segments T2–T5 eliminated the release of SP during subsequent CoAO (23). This led us to propose that focal ischemia activated cardiac ischemic-sensitive afferent neurons with subsequent neuronal release of SP into the thoracic cord, and such release was a fundamental first step in information signal processing of the ischemic event and ultimately in the perception of angina (23).

This present study indicates that there is a basal release of irDyn A-(1–13) from a broad area of the thoracic spinal cord at T4. Other studies also report a basal release of Dyn peptides from spinal cord sites (25, 45). Our data also show that, whereas CoAO produces no change in Dyn release from the T4 spinal level by itself, activation of left vagal afferents increases its release, primarily via descending projections. Interestingly, dR exacerbates LVS-induced Dyn release at T4 and differentially shifts that release to deeper lamina of the dorsal horn. These data indicate the dynamic interactions between different levels of the neuroaxis in processing of thoracic afferent inputs. In the context of the well-recognized role of opiates in modulation of pain perception (45, 47, 53), these data suggest that regulation of neural release of Dyn within the spinal cord is multifaceted, and, as demonstrated in the present study, such release may have profound effects on information transfer of primary visceral nociceptive sensory inputs.

Stimulation of the central end of the left vagus inhibited the basal release of SP from sites in the T4 spinal cord in laminae III-VII and blunted the release of SP when CoAO was applied during this stimulation. Electrophysiological studies have shown that chemical or electrical stimulation of vagal afferent fibers increases activity in a subset of neurons in the C1–C2 spinal segments (35, 40). It is also known that vagal afferent neuron stimulation decreases the electrical activity of upper thoracic spinal neurons (1). Foreman et al. (1, 18) have proposed that the integration that occurs within this C1–C2 spinal relay accounts, in part, for the vagal inhibition of the thoracic neurons. For example, disruption of the circuitry in the C1–C2 relay by the excitotoxin, ibotenic acid, eliminated the inhibition of vagal stimulation on thoracic spinal neurons (56). The results from these experiments (56) and others from Foreman's laboratory group (6, 7, 56) indicate that the inhibitory effects on thoracic neurons by vagal stimulation do not require direct descending pathways from supraspinal nuclei but rather that propriospinal nuclei in the upper cervical spinal cord are excited by vagal stimulation, and the excitation of this pathway accounts for a large part of the suppression of thoracic nuclei activity. Given these findings from Foreman's group and our laboratory, suppression of the irSP release, both at rest and during CoAO while LVS was applied, may also involve propriospinal pathways activated in the cervical segments.

It is well documented that a major source, although not the exclusive source, of SP in the superficial laminae of the spinal cord comes from primary afferent endings (3, 31, 46), and previous studies involving activation of cardiac ischemic-sensitive afferent neurons and the effects of thoracic dR support this concept (23). SP containing intrinsic interneurons (segmental and intersegmental) can also serve as the source of the SP measured with the antibody microprobes. Because LVS alone inhibited the basal resting release of irSP from laminae III-VII, the source of the resting SP could have come from intrinsic interneurons.

Left vagal afferent stimulation not only may excite cells directly in the upper cervical spinal cord, but may also activate supraspinal pathways that contribute to the inhibition of the thoracic dorsal horn sites involved with the mediation of the cardiac ischemic-sensitive signal. Ren et al. (43) suggest that propriospinal neurons in the upper cervical spinal segments are excited from a subcoeruleus/parabrachial pathway. Neurons in this pathway, in turn, are activated by vagal stimulation. Alternately, because it is well documented that vagal afferent information synapses directly into the NTS, some of the inhibitory effects of vagal stimulation may be due to activation of descending pathways from the NTS to the cervical spinal cord (16, 38). The NTS has a high concentration of SP immunoreactivity (48), and projections from NTS sites via relays, either through the pons or rostroventrolateral medulla (42, 43), may transmit an inhibitory signal to the spinal cord.

Our data here show that there is a basal release of irDyn A-(1–13), as has been reported previously (25, 45). We report herein that irDyn A release is increased by vagal stimulation from laminae I-II and from laminae IV-VI. Whereas coronary occlusion itself did not elicit the release of irDyn, coronary occlusion together with vagal stimulation caused an increase in the release of Dyn from these same laminae, an effect that was clearly the result of the LVS. dR of the afferents entering T2–T5 had no effect on the sites of basal release of this irDyn, yet it did differentially augment irDyn release toward deeper lamina in the T4 segment. Taken together, these data suggest that the source of irDyn released in response to vagal stimulation is either from higher spinal segments (possibly C1–C3 via propriospinal descending pathways) or from supraspinal sites. This finding supports previous studies that showed that most of Dyn immunoreactivity occurred in spinal neurons, with a portion derived from primary afferent fibers (36, 49), and that dR did not alter spinal cord irDyn levels in rats (5). When comparing the binding profiles of the SP-antibody microprobes during CoAO and CoAO together with vagal stimulation, these same areas (lamina I-II and IV-VI) appear to be the site of difference between the two conditions.

Our findings demonstrated a functional correlation between Dyn release and inhibition of SP release. Although we cannot, at this point, exclude the possibility that other opiates may be involved with the attenuation of SP release, we do know that endomorphin-2 was not released in response to either coronary occlusion or vagal afferent stimulation. Thus we now suggest that the increase in release of Dyn A-(1–13) at the thoracic level by vagal stimulation may be, in part or wholly, responsible for the inhibition of SP release. Although the concept that opioids reduce transmitter release from nociceptive primary afferents is not new, especially with regard to SP (20, 26, 37, 51–53), the data we present here are the first evidence that the interaction between the two peptides may account for the attenuation of the noxious signal activated by coronary occlusion during vagal stimulation.

In summary, these data suggest that left vagal afferent-mediated electrostimulatory therapy induces its analgesic effect, in part, via inhibition of SP release within the thoracic cord and that such inhibition involves a Dyn-mediated effect on the spinal cord circuits processing primary cardiac afferent neuronal inputs. Correspondingly, left vagal afferent stimulation blunted the autonomic reflexes accompanying transient CoAO. These data indicate the interdependent interactions of peripheral and central (spinal and supraspinal) neural mechanisms in the integrated response to myocardial ischemia and its pathological consequences.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by American Heart Association Grant 0151102B.


   ACKNOWLEDGMENTS
 
The authors thank Dr. K. Singh for comments regarding this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Williams, Dept. of Physiology, Quillen College of Medicine, East Tennessee State Univ., P.O. Box 70576, Stanton-Gerber Hall B-137, Johnson City, TN 37614–1708 (E-mail: williams{at}mail.etsu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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