Spinal nociceptive transmission in the spontaneously hypertensive and Wistar-Kyoto normotensive rat

Pain, 58 (1994) 169-183 0 1994 Elsevier Science

169 B.V. All rights reserved

0304-3959/94/$07.00

PAIN 2537

Spinal nociceptive transmission in the spontaneously and Wistar-Kyoto normotensive rat Alan Randich

hypertensive

* and Joel D. Robertson

Department of Psychology, Vniuersity of Alabama at Birmingham, Birmingham, AL 35294, USA (Received

14 June 1993, revision

received

4 December

1993, accepted

23 December

1993)

Summary

Background and noxious heat-evoked responses of wide-dynamic-range (WDR) and high-threshold (HT) lumbosacral spinal dorsal horn neurons were recorded in spontaneously hypertensive rats (SHRs), Wistar-Kyoto normotensive rats (WKYs), lifetime captopril-treated SHRs, SHRs with bilateral cervical vagotomy, SHRs with bilateral sino-aortic deafferentation (SAD), and SHRs with either a single or repeated administration of naloxone methobromide (NMB). Stimulus-response functions (SRFS) were generated for neurons using 15 set of heating of the foot at temperatures ranging from 38 to 52°C. Comparisons were made of neuronal response thresholds, slopes of the SRFs, mean discharge frequency during heat stimulation, arterial blood pressure (ABP), and heart rate (HR). The primary finding was that group mean SRFs for both WDR and HT neurons were shifted in a parallel, rightward fashion in SHRs compared to WKYs. Heat-evoked response thresholds were increased and asymptotic discharge frequencies were decreased in WDR and HT neurons of SHRs compared to WKYs. Analyses of group mean SRFs for WDR and HT neurons of SHRs receiving lifetime captopril treatment indicated they were normalized to the SRFs of WKYs, but detailed comparisons using discharge frequency during heat stimulation revealed that this was due to a statistical averaging effect. Specifically, lifetime captopril-treated SHRs not only showed enhanced neuronal responses to the onset of noxious heat but also enhanced adaptation of neuronal responses with continued heating compared to WKYs. Bilateral SAD in SHRs significantly increased the total discharge frequency of WDR neurons to heat stimuli between 44 and 52°C but produced no change in the response threshold for heat-evoked activation of these neurons. A similar effect of SAD was observed in HT neurons of SHRs, but the greater response thresholds of HT neurons precluded detection of any significant effect. Bilateral cervical vagotomy did not affect response thresholds, slopes, or total discharge frequencies of SHRs, although only WDR neurons were studied. SRFs of WDR and HT neurons in SHRs obtained pre- and post-administration of a single dose of NMB did not differ. However, repeated administration of NMB in SHRs resulted in a parallel, leftward shift in SRFs of both WDR and HT neurons. In all strains and treatments studied, there were no significant differences in background activities of these neurons that might contribute to the observed outcomes. In conclusion, the hypoalgesia reported in human essential hypertensives and animals with chronic hypertension may be due to a significant attenuation in spinal nociceptive transmission. Our data are not definitive on whether increased ABP produced the attenuation of spinal nociceptive transmission in SHRs because the use of the lifetime captopril treatment to lower systemic ABP of SHRs to levels of WKYs may also have affected endogenous facilitatory and inhibitory opioid systems. However, it was possible to demonstrate an inhibitory influence of both sino-aortic afferents and endogenous central opioids on spinal nociceptive transmission in the SHR. Key words: Pain; Spontaneously

hypertensive

rat; Wistar-Kyoto

* Corresponding author: Dr. Alan Randich, Department of Psychology, University of Alabama at Birmingham, Birmingham, Al 35294, USA. Tel.: (l-205) 934-9649; FAX: (l-205) 975-6110. SSDI 0304-3959(94)00013-5

normotensive

rat; Spinal neuron; (Rat)

170

Introduction

Chronic increased arterial blood pressure (ABP) or hypertension is associated with a reduced sensitivity to painful stimuli (for reviews see Randich and Maixner 1984a; Zamir and Maixner 1986; Maixner 1991; Randich and Thurston 1991). Humans with essential hypertension manifest increased sensory and pain thresholds to electrical stimulation of tooth pulp compared to normotensive controls, and significant positive correlations are observed between mean ABP and both of these thresholds (Zamir and Shuber 1980; Ghione et al. 1985, 1988). Human essential hypertensives also show increased pain thresholds and pain tolerance to noxious thermal stimulation when compared to normotensive controls (Maixner 1989, 1991). Even normotensive humans show a significant negative correlation between resting systolic ABP and VAS pain intensity ratings of a noxious mechanical stimulus (Bruehl et al. 1992). Rats with experimental chronic hypertension (2 kidney, 1 clip, Grollman wrap, DOCA-salt), genetic chronic hypertension (SHRS; Sabra H strain), or chronic hypertension produced in WKYs by hypothalamic grafts from SHRs manifest hypoalgesia compared to normotensive controls to noxious thermal stimulation in the hot-plate assay (Zamir and Segal 1979; Zamir et al. 1980; Wendel and Bennett 1981; Maixner et al. 1982; Randich 1982; Sitsen and de Jong 1983; 1984; Tsai and Lin 1987; Elam et al. 1991), noxious radiant or thermal heat in the tail-flick assay (Saavedra 1981; Wendel and Bennett 1981; Tchakarov et al. 19851 or noxious mechanical stimulation in the paw-pinch assay (Zamir and Segal 1979; Zamir et al. 1980; Chipkin and Latranyi 1984). There have been some failures to detect hypoalgesia in rats with chronic hypertension, but these have occurred with renal and DOCA-salt models for which both positive and negative outcomes have been reported (Zamir and Segal 1979; Zamir et al. 1980; Sitsen and de Jong 1983, 1984; Tsai and Lin 1987). The hypoalgesia observed in chronic hypertension has been related to alterations in opioid, cholinergic, and noradrenergic function. In all studies reported to date, systemic administration of the opioid receptor antagonist naloxone reverses the hypoalgesia manifested by SHRs in either hot-plate (Zamir and Segal 1979; Zamir et al. 1980; Saavedra 1981; Wendel and Bennett 1981; Maixner et al. 1982; Sitsen and de Jong 1983; Chipkin and Latranyi 1984; Tchakarov et al. 1985) or tail-flick assays (Chipkin and Latranyi 1984; Tchakarov et al. 1985). Systemic administration of the centrally acting muscarinic cholinergic receptor antagonist atropine sulphate, but not the peripherally acting cholinergic receptor antagonist methyl atropine significantly attenuates the hypoalgesia manifested by SHRs in the hot-plate assay (Wendel and Bennett 1981).

Administration of the cr,-adrenergic receptor antagonist yohimbine reverses the hypoalgesia in SHRs in the paw-pinch assay (Chipkin and Latranyi 1984). One possible account of the hypoalgesia observed in the chronic hypertensive organism is that increased systemic ABP acts via sino-aortic afferents to alter supraspinal and/or spinopetal pain modulatory systems. Indirect support for the “sin0-aortic” view derives from demonstrations that acute activation of carotid sinus baroreceptors by negative external cervical pressure in normotensive humans produces a decrease in pain sensitivity to electrical stimulation of tooth pulp (Kniffki et al. 1989), pressure applied to a finger (Kniffki et al. 19891, and various chronic pain syndromes (Herbert et al. 1990). Similarly, acute increases in ABP in animals produced by either pharmacological or mechanical treatments results in hypoalgesia that depends on the integrity of sino-aortic afferents (Dworkin et al. 1979; Thurston and Randich 1990). However, bilateral sino-aortic deafferentation (SAD) does not affect the hypoalgesia manifested by SHRs in the hot-plate assay (Maixner et al. 1982; Randich 1982). Further, the characteristics of sino-aortic baroreceptor resetting, which occur under conditions of chronic hypertension, would not seem conducive to the production of hypoalgesia, although caution is warranted since sino-aortic baroreceptor resetting has been studied only with respect to reflex modulation of the circulation. A second possibility- is that alterations in the lowpressure side of the circulation under conditions of chronic hypertension result in activation of cardiopulmonary vagal afferents to alter supraspinal and/or spinopetal pain modulatory systems. Indirect support for the “vagal” view derives from demonstrations that acute electrical, chemical, or physiological activation of vagal afferents inhibits both behavioral and spinal neuronal nociception in normotensive rats (for review Randich and Gebhart $992). Maixner et al. (1982) also reported that unilateral resection of the right cervical vagus resulted in a time-dependent reversal of the hypoalgesia exhibited by SHRs in the hot-plate assay. Vagal afferent involvement in the hypoalgesia manifested by the hypertensive organism is also plausible in terms of cardiopulmonary vagal baroreceptor resetting (see Maixner et al. 1982 for commentary). A corollary to this view is that under conditions of chronic hypertension there may be release of a substance that acts via vagal afferents to produce hypoalgesia. For example, intravenous administration of opioids have been demonstrated to produce inhibition of both behavioral and spinal neuronal nociception by activation of cardiopulmonary vagal afferents (Randich and Maixner 1984b; Randich et al. 1991, 1992; Randich and Gebhart 1992). A final consideration is that neither systemic ABP, systemic release of a chemical, sino-aortic afferents,

171

nor cardiopulmonary vagal afferents are important for the hypoalgesia manifested by the chronic hypertensive organism. That is, the hypoalgesia may be related to some factor other than peripheral and central mechanisms involved in the control of the circulation. In point of fact, there is a limited amount of data concerning the physiological substrates that mediate the hypoalgesia manifested by the hypertensive organism. All previous studies have used behavioral measures of nociception and a single parameter of nociceptive stimulation. The difficulty with behavioral measures of nociception is that they do not distinguish between inhibition of sensory and motor function. Similarly, the use of a single parameter of noxious stimulation provides very little information about the characteristics of nociceptive processing in the chronic hypertensive organism. Therefore, it is important to parametrically evaluate spinal neuronal processing of noxious sensory input using a selective, natural noxious stimulus that produces the sensation of pain in humans. The present experiments examined these issues by recording background and noxious heat-evoked responses of lumbosacral spinal dorsal horn neurons in SHRs and WKYs. In all experiments, stimulus-responses functions (SRFs) were generated using a range of noxious heat stimuli (38-52°C in 2°C increments). In addition, lifetime captopril-treated SHRs, bilateral cervical vagotomized SHRs, sino-aortic denervated SHRs, and SHRs treated with the opioid-receptor antagonist naloxone methobromide were evaluated to assess possible substrates for altered spinal nociceptive transmission observed in SHRs.

Methods Subjects Male SHRs and WKYs (Harlan, Pratville, AL) served as subjects. SHRs and WKYs were derived originally from the NIH strain. The rats were housed in plastic cages under a 12-h light-dark cycle. Food and water were available on an ad libitum basis. Lifetime captopril-treated SHRs were obtained from the same supplier and treated in the following manner. At the time of breeding, female SHRs were given captopril in their drinking water at a dose of 100 mg/kg/day. They were maintained on this dosage throughout pregnancy and lactation. After weaning, the pups were maintained on 50 mg/kg/day captopril in their drinking water until they were used for the present studies. The mean (+S.E.M.) ages in weeks and group designations of animals used in these studies were SHR (20.36f 1.211, WKY (18.34 + 1.671, SHR-CAPTROPRIL (24.06 + 0.39), SHR-VAGOTOMY (25.50 f 1.261, and SHR-SAD (23.31+ 0.64).

Apparatus Tungsten microelectrodes (0.8-0.95 MR; Micro-Probe, Clarksburgh, MD) were used for conventional extracellular recordings of single neurons in the La-L, spinal segments. Radiant heat from a

feedback-controlled projector lamp was focused on the glabrous skin within the unit’s receptive field. Radiant heat was held at a base temperature of 35-36°C during intertrial intervals. A copper-constantan thermocouple (ANSI type T, 0.13 mm diameter; Omega Engineering, Stamford, CT) placed in the center of the field of heat stimulation allowed for control of the temperature at the air-skin interface. Unit potentials were amplified, displayed on an oscilloscope, and isolated using an analog delay circuit (BAK DDIS-1). Unit responses were counted and saved by a computer. ABP and heart rate (HR) were continuously recorded on Epson computers from the signal provided by a Cobe pressure transducer. HR was obtained by conversion of interbeat intervals derived from the pulse pressure signal.

General surgical techniques Each rat initially received intraperitoneal administration of 50 mg/kg of pentobarbital sodium. Catheters were inserted into the trachea for artificial ventilation, left femoral artery for recording of ABP, and left femoral vein for continuous administration of anesthesia. ABP and HR were recorded at this time for an initial assessment of differences between strains and treatments. The head of the rat was then fixed in a stereotaxic apparatus and the lumbar spinal cord exposed by a laminectomy between the T,, and L, vertebrae. The spinal column was suspended by vertebral clamps rostra1 and caudal to the laminectomy, and a pool for agar (1.75% in saline) was made to minimize respiratory movements of the spinal cord. The dura mater was carefully cut and a protective bath of warm mineral oil was formed in the agar. The left hind foot was placed in a paraffin wax model with the plantar surface upward. The rats were then paralyzed with pancuronium bromide (0.4 mg i.v. initially and 0.2-0.4 mg/h thereafter), artificially ventilated, body temperature maintained with a heating pad set at 37°C and end-expired CO* was monitored and maintained between 3.5 and 4.5%. Anesthesia was maintained by a continuous i.v. infusion of pentobarbital sodium (2.54-5.10 mg/h).

General procedures Non-noxious brush and noxious pinch were applied to the hindfoot as initial search stimuli for spinal dorsal horn neurons. Neurons that responded to noxious pinch were isolated and tested for responses to noxious heating of the hindfoot (52°C 15 set duration). Any unit that responded to 15 set of noxious heating of the hindpaw at 52°C with less than 10% variation during 3 consecutive heat control trials was evaluated further and the receptive field for each unit was characterized on a figurine. SRFs were then generated by presenting heat stimuli of 15 set duration ranging in temperature from 38 to 52°C in 2°C increments. The sequence of heat stimulus presentation was generally 52, 52, 52, 48, 44, 40, 38, 50, 46, 42, and 52°C. but the lowest temperature studied varied depending on the threshold of the neuron. The intertrial interval was 3 min. Responses to repeated presentation of heat at this interval have been demonstrated to be stable for several hours. The focus of these experiments was on wide-dynamic-range (WDR; class 2) and high-threshold (HT; class 3) neurons. Units that responded to non-noxious brushing or light touch, noxious pinch and noxious heat were classified as WDR neurons. Units that responded only to noxious pinch and noxious heat were classified as HT neurons. Units that responded to a sharp tap of the foot, noxious pinch, and noxious heat, but not to non-noxious brushing or light touch were also classified as HT neurons. In most experiments, only the last neuron studied in a rat was electrolytically lesioned in order to prevent spinal cord damage, but in some experiments all neurons that were studied in a rat were lesioned.

172

Experiment I In Experiment 1, SRFs for WDR and HT spinal neurons WKY, and SHR-CAPTOPRIL were determined.

of SHR,

Experiment 2 In Experiment 2, SRFs were generated for SHR-VAGOTOMY and SHR-SAD. These SRFs were compared with the SRFs generated for the SHR control group evaluated in Experiment 1. Bilateral cervical vagotomy involved transection of the cervical vago-aortic trunk. Bilateral SAD involved stripping the carotid sinus region of all fibers, painting the region with a 10% phenol solution, and transection of the sympathetic chains, superior laryngeal nerves and aortic depressor nerves. Each operation was performed approximately 2 h prior to the initiation of the spinal recording. SAD was verified at the termination of each experiment by the lack of reflex bradycardia produced by i.v. administration of phenylephrine in a dose that produced a 60-80 mm Hg increase in ABP.

Experiment 3 In Experiment 3, SRFs for individual neurons were generated for SHRs both before and after iv. administration of 5 mg/kg of naloxone methobromide (NMBI. This procedure provided withinsubject data on the effects of NMB and we viewed this manipulation

as a test of possible peripheral opioid mediation of the alterations in spinal nociceptive transmission manifested by SHRs in Experiment 1. The generation of the SRFs were as described above and the post-NMB SRF was initiated 3 min following administration of NMB. Following the initial assessment with NMB in any given rat, 2-3 supplemental doses of NMB (S mg/kg per dose) were administered and other spinal neurons were assessed. This procedure provided for between-subject comparisons with the SHR control data obtained in Experiment I. However, we viewed these data as ambiguous with respect to either peripheral or central actions of NMB given the time and dose considerations, and are discussed only in this context.

Cardior~ascular recording As noted previously, ABP and HR were first recorded following the insertion of catheters and prior to administration of deep anesthesia and pancuronium bromide. ABP and HR were also continuously recorded during the generation of SRFs while the rats were deeply anesthetized. ABP measurements reported in the SRF data analyses were obtained 1 set prior to the onset of the heat stimuli.

Drugs Drugs used in these experiments included NMB administered a volume of S mg/ml through the external jugular catheter,

in

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Fig. 1. SRFs for WDR neurons to graded heating of the foot and ABP. (top panels) Individual SRFs for WDR neurons of groups WKY tn = 241, SHR (n = 24J and SHR-CAPTOPRIL (n = 1.5). Data are plotted as total discharges during 15 set of skin heating at the various temperatures. (bottom left panel) Group mean SRFs. Group mean regression lines were calculated from regression lines determined for each individual neuron presented in the upper panels. Group mean total discharges t+ S.E.M.) are also presented at each temperature, but were not used in the determining the mean regression lines. (lower right panel) Group mean ABP (+_S.E.M.) recorded I set prior to the onset of the heat stimuli at the various temperatures.

173

Histology

temperature. ANOVAs were performed on response thresholds and slopes derived from these analyses. Group mean discharges were used in some analyses when response threshold was unaffected by a treatment. Set-by-see analyses of group mean discharge frequencies during 15 set of heating were also performed. All ABP and HR values were expressed as a percentage change from the appropriate baseline value. Ah data were analyzed with ANOVA. Alpha was 0.05 in all analyses unless otherwise noted.

Rats were overdosed with pentobarbital sodium at the termination of an experiment. Electrolytic lesions of the recording sites were histologically identified in tissue stained with cresyl violet. As noted previously, in most experiments lesions were only made of the last neuron studied in a rat to prevent spinal cord damage, but in latter experiments, all sites of recording were lesioned and mapped according to the atlas of Paxinos and Watson (1986).

Data analysis The responses to noxious heat stimuli were quantified using a regression analysis for each rat. The only restrictions of this analysis were that the greatest absolute number of discharges obtained at either 50°C or 52°C was used as the uppermost value in the regression analysis and the lowest temperature used in the analysis was that resulting in a neuronal discharge of less than 10% of the maximum absolute discharge of the neuron at either 50°C or 52°C. These restrictions were applied to ensure that only the linear portions of the SRFs were analyzed. A mean regression line for each group was then calculated using the individual regression lines determined from each rat. This measure was derived from the individual regression lines instead of from group mean discharges because it minimizes the influence of a deviant response obtained from a single rat at any given temperature. However, these mean regression lines were plotted in conjunction with the group mean discharges during 1.5set of heating at each

Results Experiment 1: spinal nociceptive transmission in SHR, WKY, and SHR-CAPTOPRIL WDR neurons. The upper panels of Fig. 1 present

individual SRFs of WDR neurons for groups SHR (n = 24 units), WKY (n = 24 units) and SHRCAPTOPRIL (n = 15 units). The lower left panel of Fig. 1 presents the group mean SRFs for these WDR neurons. The lower left panel of Fig. 1 shows that the group mean SRF for WDR neurons of group SHR is shifted in a parallel, rightward fashion to the SRFs of both

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Fig. 2. Analyses of group mean discharges/set during 15 set of heat stimulation at the various temperatures for all WDR neurons studied in groups SHR, WKY, and SHR-CAPTOPRIL (top panels) and SHR-VAGOTOMY, SHR-SAD, and SHR-NMB (bottom panels).

groups WKY and SHR-CAPTOPRIL, which were similar. The mean response thresholds for activation of these WDR neurons were 39.34, 39.34, and 41.82”C in groups WKY, SHR-CAPTOPRIL, and SHR, respectively. The response thresholds for groups WKY and SHR-CAPTOPRIL did not significantly differ, but both of these response thresholds were significantly less than the response threshold of group SHR. The slopes of the SRFs did not significantly differ between any of these groups. The upper panels of Fig. 2 provide an analysis of group mean discharges/set of these WDR neurons both 5 set prior to and during the 15 set of the noxious heat stimuli for these groups. The bottom panels present data that will be analyzed and discussed later, but are presented in this figure for ease of comparison. Fig. 2 shows that WDR neurons of group SHR show a gradual increase in mean discharge frequency following the onset of the heat stimuli with very little frequency adaptation during continued heating. WDR neurons of group WKY discharge at a frequency approximately 50% greater than those of group SHR across all temperatures tested. Further, with the 50°C and 52°C heat stimuli, WDR neurons of WKYs tend to show an abrupt increase in mean discharge frequency following heat onset which is then followed by some adaptation during continued heating. WDR neurons of group SHR-CAPTOPRIL show response profiles that are

TABLE MEAN

I BACKGROUND

Data are expressed Group

more similar to WDR neurons of group WKY than to WDR neurons of group SHR, in that there is an abrupt increase in mean discharge frequency following the onset of the heat stimuli. However, there appears to be a greater adaptation in discharge frequency during continued heating in group SHR-CAPTOPRIL compared to group WKY. That is, adaptation occurred at temperatures ranging from 44 to 52°C in group SHR-CAPTOPRIL, but was only apparent with the 50-52°C stimuli in group WKY. Table I presents background activities of all units. There were no significant differences in the background activities of WDR neurons of groups SHR, WKY, and SHR-CAPTOPRIL prior to any temperature tested, although background activities of WDR neurons of group SHR tended to be less than those of either groups WKY or SHR-CAPTOPRIL. HT neurons. Fig. 3 presents the data for the HT neurons of these groups in a manner identical to Fig. 1. The lower left panel of Fig. 3 shows that the mean SRF for HT neurons of group SHR is also shifted in a parallel, rightward fashion compared to the SRFs of either groups WKY or SHR-CAPTOPRIL. The mean response thresholds for activation of these HT neurons were 42.97, 42.88, and 45.59”C in groups WKY (n = 14 units), SHR-CAPTOPRIL (n = 7 units), and SHR (n = 9 units), respectively. The response thresholds for HT neurons of groups WKY and SHR-CAPTOPRIL did

ACTIVITY

as discharges/set

OF UNITS

TEMPERATURES

(+ SEM)

52°C

Wide-dynamic-range neurons SHR 2.28 (n = 24) WKY 5.03 (n = 24) SHR-CAPTOPRIL 4.76 (n = 15) SHR-VAGOTOMY 5.34 (n = 14) SHR-SAD 4.65 (n = 17) SHR-NMB 9.07 (n = 12) High-threshold neurons WKY 1.45 (n = 14) SHR 3.03 (n = 9) SHR-CAPTOPRIL 0.29 (n = 7) SHR-SAD 2.15 (n = 12) SHR-NMB 0.33 (n=6)

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175

not significantly differ, but both of these thresholds were significantly less than the response threshold of group SHR. The slopes of the SRFs for HT neurons did not significantly differ between any groups. The SRFs presented in Fig. 3 give the impression that the SRF of HT neurons of group SHRCAPTOPRIL was normalized to that of group WKY. However, the analysis of mean discharges/set of HT neurons during heat stimulation shown in the upper panels of Fig. 4 suggest that this is not the case. HT neurons of group SHR show a gradual increase in mean discharge frequency following the onset of the heat stimuli with very little adaptation during continued heating, a response profile similar to those obtained for WDR neurons of group SHR. Indeed, the terminal discharge frequency at 15 set of heating is similar in WDR and HT neurons of SHR, but HT neurons require a longer time to achieve this discharge frequency because of a greater response threshold. HT neurons of group WKY show a greater number of discharges following the onset of the heat stimuli than the HT neurons of group SHR and also show little

adaptation during continued heating. However, at 15 set of heating, the maximal discharge frequencies of HT neurons are not markedly different between groups SHR and WKY. HT neurons of group SHRCAPTOPRIL, however, show a markedly different response profile than HT neurons of group WKY despite the fact that the mean SRFs presented in Fig. 3 give the impression that the two groups were similar. Specifically, HT neurons of group SHR-CAPTOPRIL show an abrupt increase in activity following the onset of heat stimulation that is greater than that observed in HT neurons of either groups SHR or WKY. Discharge frequency then dramatically adapts to levels that are either comparable to or even less than HT neurons of group SHR during continued heating at temperatures ranging from 46°C to 52°C. The background activities for these HT neurons are presented in Table I and there were no significant differences in these values between any of the groups. ABP Group mean ABP and HR obtained after initial pentobarbital anesthesia and catheterization, but prior to continuous administration of deep anesthesia,

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Fig. 3. SRFs for HT neurons to graded heating of the foot and ABP. (top panels) Individual SRFs for HT neurons of groups WKY (n = 14), SHR (n = 9), and SHR-CAPTOPRIL (n = 7). Data are plotted as total discharges during 15 set of skin heating at the various temperatures. (bottom left panel) Group mean SRFs. The group mean regression lines were calculated from regression lines determined for each individual neuron presented in the upper panels. Group mean total discharges (&S.E.M.) are also presented at each temperature, but were not used in the determining the regression lines. (lower right panel) Group mean ABP (fS.E.M.) recorded 1 set prior to the onset of the heat stimuli at the various temperatures.

176 TABLE

cantly less than either groups SHR or SHRCAPTOPRIL, which did not differ. Group mean ABP obtained 1 set prior to tests of noxious heat stimuli are presented in the lower right panels of Figs. 1 and 3 for WDR and HT neurons, respectively. For WDR neurons, there were no significant differences in mean ABP between groups WKY and SHR-CAPTOPRIL, and both of these groups had significantly lower mean ABP than group SHR at all temperatures tested. For HT neurons, the mean ABP of group WKY was significantly less than that of group SHR at all temperatures tested and did not differ from that of group SHR-CAPTOPRIL. However, the mean ABPs of group SHR-CAPTOPRIL were significantly less than those of group SHR only at the 52°C and 46°C temperatures.

II

GROUP MEAN ARTERIAL BLOOD PRESSURE AND HEART RATE (*SEMI AFTER INITIAL CATHETERIZATION, BUT PRIOR TO DEEP ANESTHESIA, PARALYSIS, AND LAMINECI’OMY Group

SHR WKY SHR-CAPTOPRIL SHR-VAGOTOMY SHR-SAD

ABP

HR

(mm Hg)

(bpm)

176.35 f 4.94 91.54 f 3.81 1X56+5.34 166.60 + 3.58 165.58 + 8.43

380.54 + 15.00 274.22 f 12.50 341.00~12.12 368.50 + 7.73 404.25 f 7.12

paralysis, laminectomy, and neuronal recordings are presented in Table II. Group SHR had a significantly greater mean ABP than either groups WKY or SHR-CAPTOPRIL. However, group SHR-CAPTOPRIL also had a significantly greater mean ABP than group WKY prior to deep anesthesia. The mean HR of group WKY was signifi-

Experiment 2: spinal nociceptive transmission in SHRVAGOTOMY and SHR-SAD WDR Neurons The upper panels of Fig. 5 present

individual SRFs of WDR neurons

for groups SHR

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of group mean discharges/set during 15 set of heat stimulation at the various temperatures for all HT neurons SHR, WKY, and SHR-CAPTOPRIL (top panels) and SHR-SAD and SHR-NMB (bottom panels).

studied

in groups

177

(reproduced from Fig. 1; n = 24 units), SHR-VAGOTOMY (n = 14 units), and SHR-SAD (n = 17 units). The lower left panel of Fig. 5 presents group mean SRFs for these WDR neurons. There were no significant differences in the mean response thresholds for activation of these WDR neurons which were 40.98, 42.08, and 41.82”C in groups SHR-SAD, SHR-VAGOTOMY, and SHR, respectively. ANOVAs were then performed on discharge frequencies at each temperature. The discharge frequency of groups SHR and SHR-VAGOTOMY did not differ at any temperature, but both of these groups showed significantly less mean discharge frequencies than group SHR-SAD at temperatures of 44-52°C. The lower left and middle panels of Fig. 2 present an analysis of group mean discharges/set of the WDR neurons for the groups SHR-VAGOTOMY and SHRSAD. The response profiles of WDR neurons of group SHR-VAGOTOMY are virtually identical to those of the WDR neurons of untreated SHRs (upper left panel) as suggested by the group mean SRF analyses. However, the WDR neurons of group SHR-SAD show little

adaptation during continued heating and tend to increase their rate of discharge during the entire period of skin heating. Table I presents background activities of all units studied and there were no significant differences between any of these groups. HT neurons. The upper panels of Fig. 6 present individual SRFs of HT neurons for groups SHR (reproduced from Fig. 3; n = 9 units > and SHR-SAD (n = 12 units). There were an insufficient number of HT neurons in group WKY-VAGOTOMY condition for analysis. The lower left panel of Fig. 6 presents group mean SRFs for these HT neurons. The mean response thresholds for activation of these HT neurons were 43.29 and 4559°C in groups SHRSAD and SHR, respectively, but there were no significant differences in these response thresholds. ANOVAs also indicated no significant differences in mean discharge frequencies at any temperatures tested. The lower left panel of Fig. 4 shows an analysis of group mean discharges/set of HT neurons in group SHR-SAD. When compared to the untreated SHRs

WDR NEURONS

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Fig. 5. SRFs for WDR neurons to graded heating of the foot and ABP. (top panels) Individual SRFs for WDR neurons of SHR (reproduced from Fig. 1; n = 24), SHR-VAGOTOMY (n = 141, and SHR-SAD (n = 171. Data are plotted as total discharges during 1.5set of skin heating at the various temperatures. (bottom left panel) Group mean SRFs. The group mean regression lines were calculated from regression lines determined for each individual neuron presented in the upper panels. Group mean total discharges (* S.E.M.) are also presented at each temperature, but were not used in the determining the regression lines. flower right panel) Group mean ABP f f S.E.M.) recorded 1 set prior to the onset of the heat stimuli at the various temperatures.

(upper Ieft panel), HT neurons of group SHR-SAD tend to show less adaptation during continued heating, not unlike that observed in WDR neurons of group SHR-SAD. It may well be the case, therefore, that the greater response thresholds on the HT neurons precludes detecting an effect of the SAD which may have emerged with longer duration heat stimulation. There were no significant differences in the background activities of these HT units (Table I). ABP Table XI indicates there were no significant between-groups differences in either mean ABP or HR prior to deep anesthesia in these groups. Group mean ABPs obtained 1 set prior to tests of the noxious heat stimuli are presented in the lower right panels of Figs. 5 and 6 for WDR and HT neurons, respectively. There were no significant differences between the mean ABPs of groups SHR, SHR-SAD, or SHR-VAGOT~MY during tests of either WDR or HT neurons.

Ex~~rn~n~ 3: spinal nociceptiue tra~missi~n in SHRNMB WDR and HT neurons. Within-subject comparisons of the effects of NMB were performed on HT (n = 6 units) and WDR (n = 8 units) neurons of SHR. There were no significant differences between the SRFs of SHRs obtained pre- and post-administration of NMB in these neurons and therefore, the data from these neurons are not presented or used in any subsequent analyses. After the initial test of a neuron with NMB, we typicaliy recorded from an additional one or two neurons. NMB was re-administered prior to each test of a neuron and produced substantial changes in heatevoked discharges of these spinal neurons. However, we can not guarantee that NMB still acted only in the periphery with repeated administration, since some animals had received as much as 15 mgfkg of NMB

HT NEURONS

TEMPERATURE

TEMPERATURE

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Fig. 6. SRFs for HT neurons to graded heating of the foot and ABP. (top panels) lndividua~ SRFs for HT neurons of SHR (reproduced from Fig. 3: n = 9) and SHR-SAD (n = 12). Data are plotted as total discharges during 15 set of skin heating at the various temperatures. (bottom left panel) Group mean SRFs. The group mean regression lines were calculated from regression lines determined for each individual neuron presented in the upper panels. Group mean total discharges (_LS.E.M.) are also presented at each temperature, but were not used in the determining the regression lines. (lower right panel) Group mean ABP (+ S.E.M.) recorded 1 set prior to the onset of the heat stimuli at the various temperatures.

179

over the course of 6-8 h. The left panel of Fig. 7 presents group mean SRFs for both WDR neurons (upper panel; n = 2 units) and HT neurons (lower panel; n = 6 units) of group SHR-NMB compared against control functions of untreated SHR from Figs. 1 and 3. The right panels present the corresponding mean ABP for these groups obtained 1 set prior to noxious heating at the various temperatures. Fig. 7 indicates that repeated administration of NMB resulted in a leftward, parallel shift in the mean SRFs of group SHR-NMB for both WDR and HT neurons. For WDR neurons, NMB treatment resulted in a significant decrease in the response threshold from 41.82”C obtained in controls to 39.86”C in SHR-NMB, but no significant change in slope. For HT neurons, NMB treatment resulted in a significant decrease in the response threshold from 4559°C obtained in controls to 41.6o”C in SHR-NMB, but no significant change in slope. The lower right panel of Fig. 2 presents an analysis of group mean discharges/set for WDR neurons of

group SHR-NMB. WDR response profiles of group SHR-NMB indicate an increased discharge frequency at all temperatures tested compared to group SHR (upper left panel). The lower right panel of Fig. 4 presenting the HT response profiles of group SHRNMB also indicates an increased discharge frequency at all temperatures tested compared to untreated SHR (upper left panel). Table I presents background activities of all units studied and there were no significant differences between groups, although WDR neurons of SHRs treated with NMB tend to show increased, but highly variable background activities. ABP Group mean ABPs obtained 1 set prior to tests of the noxious heat stimuli are presented in the right panel of Figs. 7 for WDR (upper panel) and HT (lower panel) neurons, respectively. There were no significant differences between the mean ABPs in any of the comparisons, although during testing of WDR neurons the mean ABP of group SHR-NMB tended to be slightly greater than those of group SHR.

WDR NEURONS 700

T

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Fig. 7. Group mean SRFs for WDR and HT neurons to graded heating of the skin and ABP. Group mean SRFs for WDR neurons (top left; n = 12) and HT neurons (bottom left; n = 6) of SHRs before (control) and after (NMB) administration of NMB. The group mean regression lines were calculated from regression lines determined from the SRFs of individual neurons (data not shown). Group mean total discharges (+ S.E.M.) are also presented at each temperature, but were not used in the determining the regression lines. Group mean ABP (+ S.E.M.) recorded 1 set prior to the onset of the heat stimuli at the various temperatures for WDR neurons (top right) and HT neurons (bottom right).

180

bJDR

HT

Fig. 8. Spinal recording sites of all neurons that were lesioned. WDR neurons are presented on the left side and HT neurons on the right side for ease of data presentation, but all neurons were recorded on the left side.

Recording sites. Fig. 8 presents recording sites of all neurons lesioned in these experiments. These neurons were primarily located in superficial laminae of the spinal dorsal horn, but deeper laminae were sampled as well. Depth measurements of neurons not lesioned indicated that they were in similar laminar locations as neurons that were lesioned.

Discussion

The primary goal of these studies was to characterize spinal nociceptive transmission in SHRs and WKYs. This was accomplished by recording background and noxious heat-evoked responses of WDR and HT lumbosacral spinal dorsal horn neurons. WDR and HT neurons are critical for both ascending nociceptive transmission and the generation of spinal nociceptive reflexes (Menetrey et al. 1977; Price and Dubner 1977). The present analyses showed that there is a significant attenuation of spinal nociceptive transmission in SHRs compared to WKYs as evidenced by parallel, rightward shifts in the heat-evoked SRFs of both WDR and HT neurons, i.e., an increase in response threshold and no change in slope. The analyses of spinal neuronal discharge frequencies also showed that the asymptotic levels of discharges of both WDR and HT neurons are less in SHRs as compared to WKYs. This was most apparent in the WDR neurons of SHRs, where group mean neuronal discharge frequencies were approximately 50% less than those of WKYs at a time when a stable, terminal temperatures had been achieved. In themselves, the increased response thresholds of WDR and HT neurons to noxious heat stimulation could provide an explanation for the behavioral hypoalgesia reported for the chronic hypertensive organism. For example, the greater response latencies of

SHRs compared to WKYs in tail-flick and hot-plate assays could be explained by the increased response thresholds required for heat-evoked activation of WDR and HT lumbosacral spinal dorsal horn neurons in SHRs. The reduced asymptotic levels of neuronal discharges in SHRs as compared to WKYs during exposure to a fixed-duration heat stimulus could also provide a mechanism for the increased pain tolerance to noxious thermal stimuli reported for the human essential hypertensive (Maixner 1989, 1991). There were no significant differences in background activities of WDR and HT neurons between SHRs and WKYs, although there was substantial variability in these measures. Thus, increases in response threshold and/or reductions in discharge frequency could account for reduced behavioral responses to noxious stimuli manifested by the chronic hypertensive organism. It should be noted that none of these spinal neurons were assessed for ascending projections. Therefore, our data are ambiguous with respect to whether spinal interneurons and/or spinal projection neurons demonstrate these response properties in SHRs and WKYs. A second goal of these experiments was to evaluate possible factors that contribute to differences in spinal nociceptive function between SHRs and WKYs. The most obvious factor to consider is whether increased systemic ABP is related to the reduction in spinal nociceptive function of the SHR observed in the present studies. In previous studies of hypoalgesia and chronic hypertension this issue has been vigorously debated and there is evidence both for and against the position. The most obvious data in support of this view is that systemic ABP is significantly correlated with both sensory and pain thresholds in human essential hypertensives, hypertensive animals, and even normotensive humans (Zamir and Segal 1979; Maixner et al. 1982; Ghione et al. 1985, 1988; Bruehl et al. 1992). Further, administration of hexamethonium restores both normal pain sensitivities and ABP in SHRs (Maixner et al. 1982). On the other hand, several investigators have argued against the view that hypertension produces hypoalgesia because there have been some failures to observe behavioral hypoalgesia in experimental animals models of chronic hypertension (Sitsen and de Jong 1983, 1984), human essential hypertensives and SHRs receiving administration of either antihypertensive drugs or diets continue to show behavioral hypoalgesia (Sitsen and de Jong 1984; Ghione et al. 1988), SHRs manifest increased nociceptive thresholds at an earlier age than increased ABP (Wendel and Bennett 1981; Sitsen and de Jong 1983), removal of the stenotic kidney in renal hypertensive rats significantly decreases ABP to near control levels within 5 days, but normal pain sensitivities require 15 days to return to control values (Zamir and Segal 1979), and administration of

181

systemic naloxone reverses the hypoalgesia of SHRs without affecting ABP (Zamir and Segal 1979; Zamir et al. 1980; Wendel and Bennett 1981; Maixner et al. 1982). It may well be true that systemic ABP is not casually related to the hypoalgesia. However, none of the above studies presenting data counter to the view that hypertension is producing behavioral hypoalgesia are definitive. First, the antihypertensive drug and diet treatments in the human and animal studies did not restore ABP to normotensive levels, e.g., only a 5-10 mm Hg decrease in ABP occurred in the human study of Ghione et al. (1988). Second, that SHRs develop changes in nociception prior to chronic hypertension may simply reflect differences in ability to recruit detectable changes in nociceptive as compared to cardiovascular function, and may depend more on the sensitivity of the response measure rather than on system independence. A similar argument can be advanced to account for differences in the rate of reversal of chronic hypertension and hypoalgesia in the study of renal hypertension. Third, that naloxone reversed the behavioral hypoalgesia, but not the hypertension of SHRs, suggests only that opioids are important for the maintenance of hypoalgesia, but not for the maintenance of hypertension. Unfortunately, the present data provide no definitive answer to this issue of how hypertension relates to the production of hypoalgesia. A lifetime-captopril treatment was used to reduce the ABP of SHRs to levels of WKYs in order to assess this view. In principle, if systemic ABP is causally and inversely related to spinal nociceptive transmission then the captopril treatment would be expected to “normalize” the nociceptive characteristics of WDR and/or HT neurons of the SHR to those of the WKY. The use of traditional SRF analyses of response thresholds and slopes of WDR and HT neurons suggested that the captopril treatment produced this outcome since no significant differences were observed between either the noxious heat-evoked SRFs or systemic ABPs of groups WKY and SHR-CAPTOPRIL. However, analyses of sec-byset neuronal discharge frequencies during heat stimulation did not support the view that spinal neuronal responses of group SHR-CAPTOPRIL were “normalized” to those of WKYs. Instead, HT neurons of group SHR-CAPTOPRIL showed a high frequency discharge following the onset of heat stimulation which was followed by adaptation in the frequency of discharges to levels either at or below those of untreated SHRs. This response pattern was not manifested by HT neurons of WKYs. Thus, the similarity of the mean SRFs of HT neurons of groups SHR-CAPTOPRIL and WKY really resulted from a temporal averaging of an enhanced neuronal response following heat onset with a reduced neuronal response during sustained heat stimulation. A

similar marked increase in the frequency of neuronal discharge following the onset of the heat stimulation was observed in WDR neurons of group SHRCAPTOPRIL, although a similar response profile was also observed in WKYs. However, WDR neurons of group SHR-CAPTOPRIL tended to show adaptation during continued heating at temperatures ranging from 44 to 52°C and this was only exhibited by WDR neurons of WKYs at temperatures of 50 and 52°C. It is our impression, therefore, that even though the captopril treatment in the SHR altered the response properties of SHR neurons and decreased systemic ABP to levels of WKYs, it did not necessarily normalize the response characteristics of these neurons to those of WKYs. A second way of dealing with the possible influence of chronic hypertension on spinal nociceptive transmission was to evaluate the effects of transections of carotid sinus and aortic depressor afferents. Bilateral SAD in the SHR did produce a significant increase in total discharge frequency of WDR neurons at temperatures in the noxious range (44-52”C), but did not affect the response threshold compared to untreated SHRs. A similar effect was observed in HT neurons of group SHR-SAD, although it was not statistically significant. Thus, these data demonstrate that carotid sinus and/or aortic depressor afferents exert an inhibitory effect on spinal nociceptive function in the SHR. This could account for the reduced total discharge frequency observed in SHRs compared to WKYs during heat stimulation, although some other mechanism must be responsible for differences in response threshold. A second possible mechanism noted in the introduction was that alterations in vagal afferent function of the SHR due either to changes in the low pressure side of the circulation and/or peripheral opioids that activate vagal afferents may contribute to the hypoalgesia. Maixner et al. (1982) reported that unilateral cervical vagotomy produced a time-dependent reduction in the hypoalgesia manifested by the SHR in the hot-plate assay, although unilateral vagotomized WKYs also showed a reduction in pain sensitivities. In the present studies, we were unable to find any evidence that vagal afferents and/or peripheral opioids contribute to the differences in spinal nociceptive transmission. Specifically, group mean SRFs and discharge frequencies of SHRs with acute bilateral cervical vagotomy were not different from those of untreated SHRs, although we only examined WDR neurons. We also used administration of a single dose of NMB to specifically test of the view that a peripheral opioid acting via vagal afferents contributes to these differences in neuronal responses to heat stimuli. Our previous studies with a single dose of NMB indicate that it is effective in producing peripheral, but not central blockade of opioid receptors (Randich et al. 1991, 1992). However, the SRFs of WDR and HT neurons of SHRs treated with

182

a single dose of NMB were not different pre- and post-drug. A similar negative outcome was reported by Sitsen and de Jongi4 using N-methylnaloxone in the hot-plate assay. Thus, while one other study has demonstrated a time-dependent effect of unilateral cervical vagotomy on the behavioral hypoalgesia manifested by the SHR, we were unable to find an effect of either acute bilateral cervical vagotomy or a drug treatment that might affect opioid activation of vagal afferents in the SHR. These differences with vagotomy may reflect different measures of nociceptive function (behavioral paw-lick responses in the hot-plate assay versus spinal neuronal responses to noxious heat) or more likely, may be due to differences in the delay to testing following vagotomy. Specifically, animals in the present study were tested 3-10 h following bilateral cervical vagotomy and Maixner et al. (1982) showed that the effect of unilateral cervical vagotomy in attenuating the hypoalgesia of SHRs in the hot-plate assay developed over the course of weeks. Repeated administration of NMB produced a parallel, leftward shift in the SRFs of WDR and HT neurons in SHRs. If one assumes that repeated administration of NMB resulted in a central blockade of opioid receptors (for which we have no evidence), then these data are consistent with previous studies demonstrating reversal of behavioral hypoalgesia in hypertensive rats following administration of naloxone. The discharge frequencies of WDR and HT neurons of group SHR-NMB under conditions of repeated administration of NMB were similar to those of group SHRCAPTOPRIL, yet as observed in previous studies with naloxone only the captopril treatment lowered systemic ABP. If the effects of captopril on spinal nociceptive transmission were not due to a lowering of ABP, then they may be due to the effects of captopril on metabolism of opioids. Captopril, a converting-enzyme inhibitor, prevents the hydrolysis of enkephalins by enkephalinase and also has been reported to produce analgesia (Swerts et al. 1979; Reid and Rubin 1987). Based on any simple analysis, the lifetime captopril treatment might be expected to produce even greater attenuation of spinal nociceptive transmission in the SHR as compared to the untreated SHR. However, endogenous opioids can either facilitate and/or inhibit nociceptive function. It is possible, therefore, that different endogenous opioids operating through intrinsic spinal mechanisms and/or spinopetal mechanisms differentially affect neuronal function in the SHR and mediate the effects of captopril observed in the present studies. For example, spinal cord application of low doses of the K-opioid receptor agonist U-50,488H can facilitate noxious heat-evoked responses of superficial dorsal horn neurons as evidenced by a decrease in thermal threshold and/or an increase in the number of

discharges elicited by a given thermal stimulus (Hylden et al. 1991). The thermal response profiles of the latter study were very similar to those reported for captopril-treated SHRs in the present experiments. Caudle and Isaac (1988) also observed a short-term facilitatory effect of dynorphin on C-fiber evoked reflex activity. Dynorphin-like varicosities are in apposition to ascending spinal projection neurons of the superficial dorsal horn of rat (Nahin et al. 1992). If captopril impaired metabolism of an intrinsic spinal facilitatory opioid (for which we have no evidence), such as dynorphin either acting at K receptors or influencing NMDA receptors located on WDR and HT neurons, then it might account for the augmented responses of these neurons following heat onset in group SHRCAPTOPRIL. Similarly, heat-evoked intrinsic and/or spinopetal system(s) may be concomitantly operating through spinal inhibitory opioids to attenuate spinal nociceptive transmission in the SHR. If captopril also impaired metabolism of an inhibitory opioid peptide, such as an enkephalins acting at a ~1or 6 receptors on WDR and HT neurons, then it might account for the marked adaptation of neuronal responses during continued heat stimulation observed in group SHRCAPTOPRIL. It is unclear whether this system is tonically activated since we were unable to demonstrate significant differences in background activities of these neurons between strains and treatments. Thus, this speculative view suggests that at least two mechanisms involving spinal facilitatory and inhibitory opioids may modulate spinal dorsal horn neurons and were affected by the captopril treatment. However, the data obtained with repeated administration of NMB are not necessarily consistent with this hypothesis. HT and WDR neurons of group SHR-NMB did manifest augmented heat-evoked responses during continued heating which would be consistent with blockade of an opioid inhibitory system. However, these neurons also showed strong responses immediately following heat onset which would not necessarily be consistent with NMB blockade of an opioid facilitatory system. However, these outcomes may reflect the differential affinity of NMB for p opioid versus K-opioid receptors as well as more complex and difficult issues relating to the time course for activation, relative strengths, and characteristics of interactions of these systems. In summary, these data provide new information regarding spinal nociceptive transmission in SHRs and WKYs. Spinal neuronal responses to noxious heat stimuli are attenuated in SHRs compared to WKYs. Bilateral SAD, lifetime captopril treatment and repeated administration of NMB all affected spinal nociceptive function in the SHR. These data emphasize the need to address how pain systems are affected by changes in cardiovascular function.

183

Acknowledgements This research was supported by NIH Grant 229966 to A. Randich. We wish to extend a special acknowledgement to Dr. K. Berecek for supplying the lifetime captopril-treated rats used in these experiments and Dr. G.F. Gebhart for critical comments on an earlier version of this manuscript.

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