Exaggerated cardiovascular and behavioral nociceptive responses to subcutaneous formalin in the spontaneously hypertensive rat

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Neuroscience Letters 201 (1995) 9-12

Exaggerated cardiovascular and behavioral nociceptive responses to subcutaneous formalin in the spontaneously hypertensive rat Bradley K. Taylor a,b,*,M. Alex Petersonasb, Allan I. Basbaumavb9c aW.M. Keck Foundation Centerfor Integrative Neuroscience, University of California San Francisco, Box 0452, San Francisco, CA 94143-0452, USA bDepartment of Anatomy, University of California San Francisco, San Francisco, CA 94143-0452, USA ‘Department of Physiology, University of California San Francisco, San Francisco, CA 94143-0452, USA

Received 21 July 1995; revised version received 5 October 1995; accepted 13 October 1995

Abstract Spontaneously hypertensive rats (SHRs) are typically less responsive to phasic noxious stimuli than are their normotensive controls. Here, we used the formalin test to compare behavioral and cardiovascular responses to persistent noxious stimuli. Hindpaw formalin injection produced exaggerated flinching, arterial pressure and heart rate responses in SHRs, suggesting that abnormalities in blood pressure control systems increase nociceptive responses to persistent noxious stimuli. Keywords:

Spontaneously

hypertensive

rat; Hyperalgesia;

Nociception;

Subjects with elevated blood pressure typically exhibit attenuated nociceptive responses to phasic noxious stimuli, indicating that blood pressure control systems may contribute to the processing of nociceptive information [9]. For example, genetically hypertensive rats exhibit hypoalgesia in phasic pain models, including the hot-plate test [11,19,25], the paw-pinch test [2,25], and a visceral pain test [ 131. Mean arterial pressure (MAP) in humans is also inversely correlated with the magnitude of both behavioral withdrawal reflexes and visual analog scale scores to the pain produced by tooth pulp stimulation [4] or finger pinch [l]. Other studies, however, have not reported significant differences between the spontaneously hypertensive rat (SHR) and their normotensive WistarKyoto (WKY) controls [13,19,21]. The contribution of blood pressure control systems to nociceptive response in the setting of persistent noxious stimuli, i.e. tissue injury, is also unclear [2,10,21,24]. For example, compared to normotensives, the SHR exhibits less ‘pain’ (autotomy) behavior after sciatic nerve section [24] and increased mechanical paw withdrawal and tail flick latencies after subplantar yeast injection [2]. In con* Corresponding author. Tel.: +1 415 4764311; fax: +l 415 4764845; e-mail: [email protected].

Blood pressure; Heart rate; Formalin

trast, Luo and Wiesenfeld-Hallin [lo] found that peripheral nerve injury-induced flexor reflex hyperexcitability, an index of neuropathic pain, was greater in the SHR. Also, Tchakarov et al. [21] reported that nociceptive responses to intraplantar formalin injection were not different in normotensive and hypertensive rats. In this latter study, however, the experimental design required repeated injections of formalin, and used a subjective behavioral measure of nociception. To circumvent these limitations, we evaluated objective cardiovascular and behavioral measures of nociception after a single injection of formalin into the hindpaw of SHR and WKY rats. Male SHR and WKY rats, 255-300 g (11-12 weeks old), were obtained from Charles River Laboratories (Hollister, CA). As previously described [20], animals were (a) instrumented with chronic femoral arterial catheters 3-5 days before testing; (b) acclimated to the test environment for at least 16 h; (c) restrained for 1 min; and then, about 20 min later, (d) injected S.C. with formalin (37%, w/w, formaldehyde, diluted in 0.9% saline) into the plantar surface of the right hindpaw. To calculate stimulus-evoked changes in MAP or heart rate (HR), we subtracted the average of five sequential steady-state baseline cardiovascular values (collected during the ‘pre-restraint’ period just before restraint) from post-stimulus values.

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Letters 201 (1995) 9-12

Table 1 Resting MAP and HR in different experimental groups Formalin dose

Strain

SHR

5.0% 1.25% 5.0% 1.25%

WKY

MAP (mmHg)

n

8 9 8 8

HR (bpm)

Pre-restraint

Pre-formalin

Pm-restraint

Pre-formalin

141 k 6* 154 + 4+ 117+2 117*3

143 + 6* 149+4* 117*3 116k2

300 + 12 317 f 10 323 2 8 321 + 13

318+9 321 k 10 317*5 327 + 10

Values represent group mean f SEM. II, number of animals in each group. bpm, beats per minute. *Significantly different from WKY (P < 0.05).

MAP, HR, and behavioral flinches were recorded for 70 min post-formalin. Each animal was used only once. We studied four groups using a balanced design: (1) SHR given 5.0% formalin; (2) SHR given 1.25% formalin; (3) WKY given 5.0% formalin; and (4) WKY given 1.25% formalin. Pre-restraint

and

pre-formalin

baselines

were

com-

(WKY versus SHR) by ANOVA. Restraint- and formalin-evoked cardiovascular responses during the first 10 min post-stimulus were analyzed using time (min l-10) and stimulus (restraint versus formalin) as the within-subjects factors, and concentration (1.25 versus 5.0% formalin) and strain as the between-subjects factors, whereas later formalin-evoked cardiovascular responses and all flinching responses were analyzed by three-way repeated-measures ANOVA (time X concentration x strain). If significant, we separately evaluated effects during Phase 1 (min l-5), Interphase (min 1 l-15), and Phase 2 (min 21-50), unless otherwise noted. Finally, we determined the Pearson product-moment correlation coefficients between formalin-evoked flinching, MAP, and HR responses over min l-70. Pre-restraint and pre-formalin values did not differ in any of the groups (Table l), indicating that restraintevoked increases had subsided before the formalin injection. MAP in SHRs was significantly greater than that in WKYs (P < 0.05); however, HR did not differ between the strains (P > 0.05). In SHRs, injection of 5% formalin produced more flinches than did 1.25% formalin (F( 1,15) = 7.6, P < 0.05; Fig. 1A). In contrast, the two concentrations produced similar responses in WKYs (P > 0.05). Importantly, SHRs exhibited more flinches than did WKYs during Phase 1 (F( 1,29) = 26.67, P < 0.001) and Phase 2 (F(1,29) = 21.03, P < O.OOl), but not during the Interphase (P > 0.05). Behavioral and MAP responses were less variable than HR responses. The two formalin concentrations produced similar MAP and HR responses (Fig. lB,C, respectively) in both strains (P > 0.05). Although Phase 2 appears different in the WKY groups, this did not reach statistical significance, even across timepoints 31-60 for MAP (F(1,14) = 4.35, P = 0.06), or across timepoints 16-70 for pared

across

strain

HR (F(1,14) = 2.63, P > 0.05). In Phase 1, formalin produced greater early-onset MAP and HR responses than did restraint alone ((F( 1,29) = 91.2, P < O.OOl), (F( 1,29) = 72.6, P < O.OOl), respectively). Compared to WKYs, SHRs exhibited greater restraint- and formalinevoked Phase 1 changes in MAP (F( 1,29) = 10.8, P < 0.005), but not HR (P > 0.05). This strain difference was greater after formalin than after restraint, as indicated by a (F( 1,29) = 4.9, P < 0.05). stimulus X strain interaction Thus, the pain associated with the injection (and not just the stress associated with restraint) contributed to the exaggerated formalin response in the SHR. In the Interphase, although MAP and HR gradually declined to a trough 10-15 min post-formalin in both strains, they did not reach baseline levels. Compared to WKYs, SHRs exhibited greater MAP (F( 1,29) = 7.8, P < 0.001) but not HR (P > 0.05) during the Interphase. In Phase 2, restraint did not produce late-onset MAP or HR increases in either strain (data not shown). Compared to WKYs, SHRs exhibited greater Phase 2 increases in MAP (F( 1,29) = 12.0, P < 0.005) and HR (F(1,29) = 4.3, P < 0.05). These were manifested as an increase in magnitude, not duration. Finally, Table 2 illustrates that formalin-evoked MAP, HR, and flinching responses in SHR and WKY rats were highly correlated. This strong correlation suggests that a common integration center simultaneously activated both motor and cardiovascular systems. Numerous abnormalities in the SHR may contribute to altered pain transmission. For example, animal studies using phasic nociceptive stimuli argue that the exaggerated stimulation of vagal afferents [ 15,161 or sino-aortic baroreceptor afferents [3,22] associated with hypertension more strongly activates a spinopetal noradrenergic antinociceptive system in the SHR [22]. Such abnormalities in blood pressure control systems participate in nociceptive processing prior to the development of hypertension, since 4-week-old SHRs, compared to age-matched WKYs, exhibited reduced nociceptive responses to phasic noxious stimuli [18,19]. Also, experimental induction of hypertension in genetically-normal rats does not necessarily reduce the magnitude of nociceptive responses ([19], but see [25]). Regardless, since the above studies only evaluated behavioral responses to phasic noxious

B.K. Taylor et al. /Neuroscience

Letters 201 (1995) 9-12

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Table 2 Pearson product-moment correlation coefficients between (pressor), HR (tachycardia), and behavior (flinching) responses Correlation

WKY

Pressor vs. tachycardia Pressor vs. behavior Tachycardia vs. behavior *P < 0.005,

lhraint , 0

10

F‘ormalin 0

10

20

30

40

SO

60

Time (minutes) Fig. 1. (A) Number of flinches in SHR rats after 5% (B) and 1.25% (A) formalin, and WKY rats after 5% (0) and 1.25% (A) formalin. (B,C) Changes in MAP and heart Me, respectively, following 1 min of restraint and then restraint plus intraplantar formalin injection. bpm, beats per minute. n = 8-9. Values represent mean f SEM. See text for statistically significant differences.

stimuli, it would be of interest to evaluate the contribution of vagal or baroreceptor afferents to persistent pain transmission.

MAP

SHR

1.25%

5.0%

1.25%

5.0%

0.95** 0.87** 0.83**

0.89** 0.79* 0.87**

0.92** 0.74* 0.85**

0.92** 0.85** 0.95**

**P < 0.001.

Several other mechanisms may have contributed to the exaggerated responses in the SHR. For example, Hao and Wiesenfeld-Hallin [5] found that the discharge frequency of myelinated afferents arising from a neuroma was significantly higher in SHRs than in normotensive rats, suggesting that peripheral nerve activity (i.e. that produced by formalin) may also be higher in the SHR. Since phasic thermal stimuli evoked less firing in spinal cord nociresponsive neurons in SHR compared to WKY rats [17], it would be of particular interest to compare the effects of persistent noxious stimuli (i.e. hindpaw formalin) on spinal cord neuronal activity in both strains. Second, flexor reflex hyperexcitability following sciatic nerve section is significantly greater in the SHR [lo], suggesting that exaggerated central sensitization mechanisms initiated by formalin-induced tissue injury contributed to greater Phase 2 in the SHR. Third, noxious stimuli produce greater sympatho-adrenal medullary release [ 121, hypothalamo-pituitary-adrenal release [6,7], and sympathetic activity [8,14] in the SHR, suggesting that formalinevoked hormonal release or sympathetic drive contribute to exaggerated cardiovascular responses in the SHR. Furthermore, increased sympathetic tone is associated with a greater inflammatory response [8], which would increase nociceptor activation. On the other hand, since increases during Phase 1 were greater after formalin injection than after restraint alone, and since restraint did not produce Phase 2 responses, the stress associated with restraint was probably not a major contributor to the exaggerated nociceptive responses in the SHR. Finally, the activity of supraspinal opioidergic pain controls may differ in SHR and WKY rats [11,18,19]. In summary, compared to normotensive rats, SHRs exhibit greater nociceptive responses to persistent noxious stimuli. Our results are consistent with those of Luo and Wiesenfeld-Hallin [lo], who found that SHRs exhibited greater peripheral nerve injury-induced flexor reflex hyperexcitability, but they contrast with previous studies showing reduced nociceptive responses to phasic noxious stimuli in SHRs. We suggest that abnormalities in blood pressure control systems decrease nociceptive responses to phasic noxious stimuli and increase nociceptive responses to persistent noxious stimuli. Future studies of

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B. K. Taylor et al. /Neuroscience

persistent nociception in the SHR should provide valuable information regarding the relationship between pain and hypertension in humans.

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This research was supported by grants NS21445 and DA 08377. Dr. Taylor was a postdoctoral fellow supported by Training Grant NS07265.

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