An investigation of the interrelationship between manipulative therapy-induced hypoalgesia and sympathoexcitation

Objective: To investigate a proposed model in which manipulative therapy produces a treatment-specific initial hypoalgesic and sympathoexcitatory effect by activating a descending pain inhibitory system. The a priori hypothesis tested was that manipulative therapy produces mechanical hypoalgesia and sympatho-excitation beyond that produced by placebo or control. Furthermore, these effects would be correlated, thus supporting the proposed model. Design: A randomized, double-blind, placebo-controlled, repeated-measures study of the initial effect of treatment. Setting: Clinical neurophysiology laboratory. Subjects: Twenty-four subjects (13 women and 11 men; mean age, 49 yr) with chronic lateral epicondylalgia (average duration, 6.2 months). Intervention: Cervical spine lateral glide oscillatory manipulation, placebo and control. Outcome Measures: Pressure pain threshold, thermal pain threshold, pain-free grip strength test, upper limb tension test 2b, skin conductance, pileous and glabrous skin temperature and blood flux. Results: Treatment produced hypoalgesic and sympathoexcitatory changes significantly grater than those of placebo and control (p < .03). Confirmatory factor-analysis modeling, which was performed on the pain-related measures and the indicators of sympathetic nervous system function, demonstrated a significant correlation (r = .82) between the latencies of manipulation-induced hypoalgesia and sympathoexcitation. The Lagrange Multiplier test and Wald test indicated that the two latent factors parsimoniously and appropriately represented their observed variables. Conclusions: Manual therapy produces a treatment-specific initial hypoalgesic and sympathoexcitatory effect beyond that of placebo or control. The strong correlation between hypoalgesic and sympathoexcitatory effects suggests that a central control mechanism might be activated by manipulative therapy.

Sympathoexcitation during chemoreflex active expiration is mediated by L-glutamate in the RVLM/Botzinger complex of rats

Moraes DJ, Zoccal DB, Machado BH. Sympathoexcitation during chemoreflex active expiration is mediated by L-glutamate in the RVLM/Botzinger complex of rats. J Neurophysiol 108: 610-623, 2012. First published April 25, 2012; doi:10.1152/jn.00057.2012.-The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla/Botzinger/pre-Botzinger complexes (RVLM/BotC/pre-BotC) on the respiratory modulation of sympathoexcitatory response to peripheral chemoreflex activation (chemoreflex) was evaluated in the working heart-brain stem preparation of juvenile rats. We identified different types of baro- and chemosensitive presympathetic and respiratory neurons intermingled within the RVLM/BotC/pre-BotC. Bilateral microinjections of kynurenic acid (KYN) into the rostral aspect of RVLM (RVLM/BotC) produced an additional increase in frequency of the phrenic nerve (PN: 0.38 +/- 0.02 vs. 1 +/- 0.08 Hz; P < 0.05; n = 18) and hypoglossal (HN) inspiratory response (41 +/- 2 vs. 82 +/- 2%; P < 0.05; n = 8), but decreased postinspiratory (35 +/- 3 vs. 12 +/- 2%; P < 0.05) and late-expiratory (24 +/- 4 vs. 2 +/- 1%; P < 0.05; n = 5) abdominal (AbN) responses to chemoreflex. Likewise, expiratory vagal (cVN; 67 +/- 6 vs. 40 +/- 2%; P < 0.05; n = 5) and expiratory component of sympathoexcitatory (77 +/- 8 vs. 26 +/- 5%; P < 0.05; n = 18) responses to chemoreflex were reduced after KYN microinjections into RVLM/BotC. KYN microinjected into the caudal aspect of the RVLM (RVLM/pre-BotC; n = 16) abolished inspiratory responses [PN (n = 16) and HN (n = 6)], and no changes in magnitude of sympathoexcitatory (n = 16) and expiratory (AbN and cVN; n = 10) responses to chemoreflex, producing similar and phase-locked vagal, abdominal, and sympathetic responses. We conclude that in relation to chemoreflex activation 1) ionotropic glutamate receptors in RVLM/BotC and RVLM/pre-BtC are pivotal to expiratory and inspiratory responses, respectively; and 2) activation of ionotropic glutamate receptors in RVLM/BotC is essential to the coupling of active expiration and sympathoexcitatory response.

Glutamatergic Antagonism in the NTS Decreases Post-Inspiratory Drive and Changes Phrenic and Sympathetic Coupling During Chemoreflex Activation

Costa-Silva JH, Zoccal DB, Machado BH. Glutamatergic antagonism in the NTS decreases post-inspiratory drive and changes phrenic and sympathetic coupling during chemoreflex activation. J Neurophysiol 103: 2095-2106, 2010. First published February 17, 2010; doi: 10.1152/jn.00802.2009. For a better understanding of the processing at the nucleus tractus solitarius (NTS) level of the autonomic and respiratory responses to peripheral chemoreceptor activation, herein we evaluated the role of glutamatergic neurotransmission in the intermediate (iNTS) and caudal NTS (cNTS) on baseline respiratory parameters and on chemoreflex-evoked responses using the in situ working heart-brain stem preparation (WHBP). The activities of phrenic (PND), cervical vagus (cVNA), and thoracic sympathetic (tSNA) nerves were recorded before and after bilateral microinjections of kynurenic acid (Kyn, 5 nmol/20 nl) into iNTS, cNTS, or both simultaneously. In WHBP, baseline sympathetic discharge markedly correlated with phrenic bursts (inspiration). However, most of sympathoexcitation elicited by chemoreflex activation occurred during expiration. Kyn microinjected into iNTS or into cNTS decreased the postinspiratory component of cVNA and increased the duration and frequency of PND. Kyn into iNTS produced no changes in sympathoexcitatory and tachypneic responses to peripheral chemoreflex activation, whereas into cNTS, a reduction of the sympathoexcitation, but not of the tachypnea, was observed. The pattern of phrenic and sympathetic coupling during the chemoreflex activation was an inspiratory-related rather than an expiratory-related sympathoexcitation. Kyn simultaneously into iNTS and cNTS produced a greater decrease in postinspiratory component of cVNA and increase in frequency and duration of PND and abolished the respiratory and autonomic responses to chemoreflex activation. The data show that glutamatergic neurotransmission in the iNTS and cNTS plays a tonic role on the baseline respiratory rhythm, contributes to the postinspiratory activity, and is essential to expiratory-related sympathoexcitation observed during chemoreflex activation.

Are l-glutamate and ATP cotransmitters of the peripheral chemoreflex in the rat nucleus tractus solitarius ?

Peripheral chemoreflex activation in awake rats or in the working heart-brainstem preparation (WHBP) produces sympathoexcitation, bradycardia and an increase in the frequency of phrenic nerve activity. Our focus is the neurotransmission of the sympathoexcitatory component of the chemoreflex within the nucleus of the tractus solitarius (NTS), and recently we verified that the simultaneous antagonism of ionotropic glutamate and purinergic P(2) receptors in the NTS blocked the pressor response and increased thoracic sympathetic activity in awake rats and WHBP, respectively, in response to peripheral chemoreflex activation. These previous data suggested the involvement of ATP and L-glutamate in the NTS in the processing of the sympathoexcitatory component of the chemoreflex by unknown mechanisms. For a better understanding of these mechanisms, here we used a patch-clamp approach in brainstem slices to evaluate the characteristics of the synaptic transmission of NTS neurons sending projections to the ventral medulla, which include the premotor neurons involved in the generation of the sympathetic outflow. The NTS neurons sending projections to the ventral medulla were identified by previous microinjection of the membrane tracer dye, 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate (DiI), in the ventral medulla and the spontaneous (sEPSCs) and tractus solitarius (TS)-evoked excitatory postsynaptic current (TS-eEPSCs) were recorded using patch clamp. With this approach, we made the following observations on NTS neurons projecting to the ventral medulla: (i) the sEPSCs and TS-eEPSCs of DiI-labelled NTS neurons were completely abolished by 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), an antagonist of ionotropic non-NMDA glutamatergic receptors, showing that they are mediated by L-glutamate; (ii) application of ATP increased the frequency of appearance of spontaneous glutamatergic currents, reflecting an increased exocytosis of glutamatergic vesicles; and (iii) ATP decreased the peak of TS-evoked glutamatergic currents. We conclude that L-glutamate is the main neurotransmitter of spontaneous and TS-evoked synaptic activities in the NTS neurons projecting to the ventral medulla and that ATP has a dual modulatory role on this excitatory transmission, facilitating the spontaneous glutamatergic transmission and inhibiting the TS-evoked glutamatergic transmission. These data also suggest that ATP is not acting as a cotransmitter with L-glutamate, at least at the level of this subpopulation of NTS neurons studied.

INTERMITTENT ACTIVATION OF PERIPHERAL CHEMORECEPTORS IN AWAKE RATS INDUCES Fos EXPRESSION IN ROSTRAL VENTROLATERAL MEDULLA-PROJECTING NEURONS IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS

Despite the well-established sympathoexcitation evoked by chemoreflex activation, the specific sub-regions of the CNS underlying such sympathetic responses remain to be fully characterized. In the present study we examined the effects of intermittent chemoreflex activation in awake rats on Fos-immunoreactivity (Fos-ir) in various subnuclei of the paraventricular nucleus of the hypothalamus (PVN), as well as in identified neurosecretory preautonomic PVN neurons. In response to intermittent chemoreflex activation, a significant increase in the number of Fos-ir cells was found in autonomic-related PVN subnuclei, including the posterior parvocellular, ventromedial parvocellular and dorsal-cap, but not in the neurosecretory magnocellular-containing lateral magnocellular subnucleus. No changes in Fos-ir following chemoreflex activation were observed in the anterior PVN subnucleus. Experiments combining Fos immunohistochemistry and neuronal tract tracing techniques showed a significant increase in Fos-ir in rostral ventrolateral medulla (RVLM)-projecting (PVN-RVLM), but not in nucleus of solitarii tract (NTS)-projecting PVN neurons. In summary, our results support the involvement of the PVN in the central neuronal circuitry activated in response to chemoreflex activation, and indicate that PVN-RVLM neurons constitute a neuronal substrate contributing to the sympathoexcitatory component of the chemoreflex. Published by Elsevier Ltd on behalf of IBRO.

Asymmetrical changes in lumbar sympathetic nerve activity following stimulation of the sciatic nerve in rat

Noxious stimulation of the leg increases hind limb blood flow (HBF) to the ipsilateral side and decreases to the contralateral in rat. Whether or not this asymmetrical response is due to direct control by sympathetic terminals or mediated by other factors such as local metabolism and hormones remains unclear. The aim of this study was to compare responses in lumbar sympathetic nerve activity, evoked by stimulation of the ipsilateral and contralateral sciatic nerve (SN). We also sought to determine the supraspinal mechanisms involved in the observed responses. In anesthetized and paralyzed rats, intermittent electrical stimulation (1 mA, 0.5 Hz) of the contralateral SN evoked a biphasic sympathoexcitation. Following ipsilateral SN stimulation, the response is preceded by an inhibitory potential with a latency of 50 ms (N=26). Both excitatory and inhibitory potentials are abolished following cervical Cl spinal transection (N=6) or bilateral microinjections of muscimol (N=6) in the rostral ventrolateral medulla (RVLM). This evidence is suggestive that both sympathetic potentials are supraspinally mediated in this nucleus. Blockade of RVLM glutamate receptors by microinjection of kynurenic acid (N=4) selectively abolished the excitatory potential elicited by ipsilateral SN stimulation. This study supports the physiological model that activation of hind limb nociceptors evokes a generalized sympathoexcitation, with the exception of the ipsilateral side where there is a withdrawal of sympathetic tone resulting in an increase in HBF. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.

Alpha1-adrenoceptor-dependent vascular hypertrophy and remodeling in murine hypoxic pulmonary hypertension.

Excessive proliferation of vascular wall cells underlies the development of elevated vascular resistance in hypoxic pulmonary hypertension (PH), but the responsible mechanisms remain unclear. Growth-promoting effects of catecholamines may contribute. Hypoxemia causes sympathoexcitation, and prolonged stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs) induces hypertrophy and hyperplasia of arterial smooth muscle cells and adventitial fibroblasts. Catecholamine trophic actions in arteries are enhanced when other conditions favoring growth or remodeling are present, e.g., injury or altered shear stress, in isolated pulmonary arteries from rats with hypoxic PH. The present study examined the hypothesis that catecholamines contribute to pulmonary vascular remodeling in vivo in hypoxic PH. Mice genetically deficient in norepinephrine and epinephrine production [dopamine beta-hydroxylase(-/-) (DBH(-/-))] or alpha(1)-ARs were examined for alterations in PH, cardiac hypertrophy, and vascular remodeling after 21 days exposure to normobaric 0.1 inspired oxygen fraction (Fi(O(2))). A decrease in the lumen area and an increase in the wall thickness of arteries were strongly inhibited in knockout mice (order of extent of inhibition: DBH(-/-) = alpha(1D)-AR(-/-) > alpha(1B)-AR(-/-)). Distal muscularization of small arterioles was also reduced (DBH(-/-) > alpha(1D)-AR(-/-) > alpha(1B)-AR(-/-) mice). Despite these reductions, increases in right ventricular pressure and hypertrophy were not attenuated in DBH(-/-) and alpha(1B)-AR(-/-) mice. However, hematocrit increased more in these mice, possibly as a consequence of impaired cardiovascular activation that occurs during reduction of Fi(O(2)). In contrast, in alpha(1D)-AR(-/-) mice, where hematocrit increased the same as in wild-type mice, right ventricular pressure was reduced. These data suggest that catecholamine stimulation of alpha(1B)- and alpha(1D)-ARs contributes significantly to vascular remodeling in hypoxic PH.

Angiotensin and baroreflex control of the circulation

There is a close association between the location of angiotensin (Ang) receptors and many important brain nuclei involved in the regulation of the cardiovascular system. The present review encompasses the physiological role of Ang II in the brainstem, particularly in relation to its influence on baroreflex control of the heart and kidney. Activation of AT1 receptors in the brainstem by fourth ventricle (4V) administration to conscious rabbits or local administration of Ang II into the rostral ventrolateral medulla (RVLM) of anesthetized rabbits acutely increases renal sympathetic nerve activity (RSNA) and RSNA baroreflex responses. Administration of the Ang antagonist Sarile into the RVLM of anesthetized rabbits blocked the effects of Ang II on the RSNA baroreflex, indicating that the RVLM is the major site of sympathoexcitatory action of Ang II given into the cerebrospinal fluid surrounding the brainstem. However, in conscious animals, blockade of endogenous Ang receptors in the brainstem by the 4V AT1 receptor antagonist losartan resulted in sympathoexcitation, suggesting an overall greater activity of endogenous Ang II within the sympathoinhibitory pathways. However, the RSNA response to airjet stress in conscious rabbits was markedly attenuated. While we found no effect of acute central Ang on heart rate baroreflexes, chronic 4V infusion inhibited the baroreflex and chronic losartan increased baroreflex gain. Thus, brainstem Ang II acutely alters sympathetic responses to specific afferent inputs thus forming part of a potentially important mechanism for the integration of autonomic response patterns. The sympathoexcitatory AT1 receptors appear to be activated during stress, surgery and anesthesia.

Functional mapping of the cardiorespiratory effects of dorsal and median raphe nuclei in the rat

The dorsal (DRN) and median (MRN) raphe nuclei are important sources of serotonergic innervation to the forebrain, projecting to sites involved in cardiovascular regulation. These nuclei have been mapped using electrical stimulation, which has the limitation of stimulating fibers of passage. The present study maps these areas with chemical stimulation, investigating their influence on cardiorespiratory parameters. Urethane-anesthetized (1.2 g/kg, iv) male Wistar rats (280-300 g) were instrumented for pulsatile and mean blood pressure (MBP), heart rate, renal nerve activity, and respiratory frequency recordings. Microinjections of L-glutamate (0.18 M, 50-100 nl with 1% Pontamine Sky Blue) were performed within the DRN or the MRN with glass micropipettes. At the end of the experiments the sites of microinjection were identified. The majority of sites within the MRN (86.1%) and DRN (85.4%) evoked pressor responses when stimulated (DRN: deltaMBP = +14.7 ± 1.2; MRN: deltaMBP = +13.6 ± 1.3 mmHg). The changes in renal nerve activity and respiratory rate caused by L-glutamate were +45 ± 11 and +42 ± 9% (DRN; P < 0.05%), +40 ± 10 and +29 ± 7% (MRN, P < 0.05), respectively. No significant changes were observed in saline-microinjected animals. This study shows that: a) the blood pressure increases previously observed by electrical stimulation within the raphe are due to activation of local neurons, b) this pressor effect is due to sympathoexcitation because the stimulation increased renal sympathetic activity but did not produce tachycardia, and c) the stimulation of cell bodies in these nuclei also increases the respiratory rate.

Exercise changes regional vascular control by commissural NTS in spontaneously hypertensive rats

Ogihara CA, Schoorlemmer GHM, Levada AC, Pithon-Curi TC, Curi R, Lopes OU, Colombari E, Sato MA. Exercise changes regional vascular control by commissural NTS in spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 299: R291-R297, 2010. First published April 21, 2010; doi: 10.1152/ajpregu.00055.2009.-Inhibition of the commissural nucleus of the solitary tract (commNTS) induces a fall in sympathetic nerve activity and blood pressure in spontaneously hypertensive rats (SHR), which suggests that this subnucleus of the NTS is a source of sympathoexcitation. Exercise training reduces sympathetic activity and arterial pressure. The purpose of the present study was to investigate whether the swimming exercise can modify the regional vascular responses evoked by inhibition of the commNTS neurons in SHR and normotensive Wistar-Kyoto (WKY) rats. Exercise consisted of swimming, 1 h/day, 5 days/wk for 6 wks, with a load of 2% of the body weight. The day after the last exercise session, the rats were anesthetized with intravenous alpha-chloralose, tracheostomized, and artificially ventilated. The femoral artery was cannulated for mean arterial pressure (MAP) and heart rate recordings, and Doppler flow probes were placed around the lower abdominal aorta and superior mesenteric artery. Microinjection of 50 mM GABA into the commNTS caused similar reductions in MAP in swimming and sedentary SHR (-25 +/- 6 and -30 +/- 5 mmHg, respectively), but hindlimb vascular conductance increased twofold in exercised vs. sedentary SHR (54 +/- 8 vs. 24 +/- 5%). GABA into the commNTS caused smaller reductions in MAP in swimming and sedentary WKY rats (-20 +/- 4 and -16 +/- 2 mmHg). Hindlimb conductance increased fourfold in exercised vs. sedentary WKY rats (75 +/- 2% vs. 19 +/- 3%). Therefore, our data suggest that the swimming exercise induced changes in commNTS neurons, as shown by a greater enhancement of hindlimb vasodilatation in WKY vs. SHR rats in response to GABAergic inhibition of these neurons.

Macrophage Migration Inhibitory Factor in the Paraventricular Nucleus Plays a Major Role in the Sympathoexcitatory Response to Salt

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sympatho-excitation during chemoreflex active expiration is mediated by L-glutamate in the RVLM/Bötzinger complex of rats

The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla/Bötzinger/pre-Bötzinger complexes (RVLM/BötC/pre-BötC) on the respiratory modulation of sympathoexcitatory response to peripheral chemoreflex activation (chemoreflex) was evaluated in the working heart-brain stem preparation of juvenile rats. We identified different types of baro- and chemosensitive presympathetic and respiratory neurons intermingled within the RVLM/BötC/pre-BötC. Bilateral microinjections of kynurenic acid (KYN) into the rostral aspect of RVLM (RVLM/BötC) produced an additional increase in frequency of the phrenic nerve (PN: 0.38 ± 0.02 vs. 1 ± 0.08 Hz; P < 0.05; n = 18) and hypoglossal (HN) inspiratory response (41 ± 2 vs. 82 ± 2%; P < 0.05; n = 8), but decreased postinspiratory (35 ± 3 vs. 12 ± 2%; P < 0.05) and late-expiratory (24 ± 4 vs. 2 ±1%; P < 0.05; n = 5) abdominal (AbN) responses to chemoreflex. Likewise, expiratory vagal (cVN; 67 ± 6 vs. 40 ± 2%; P < 0.05; n = 5) and expiratory component of sympathoexcitatory (77 ± 8 vs. 26 ± 5%; P < 0.05; n = 18) responses to chemoreflex were reduced after KYN microinjections into RVLM/BötC. KYN microinjected into the caudal aspect of the RVLM (RVLM/pre-BötC; n = 16) abolished inspiratory responses [PN (n = 16) and HN (n = 6)], and no changes in magnitude of sympathoexcitatory (n = 16) and expiratory (AbN and cVN; n = 10) responses to chemoreflex, producing similar and phase-locked vagal, abdominal, and sympathetic responses. We conclude that in relation to chemoreflex activation 1) ionotropic glutamate receptors in RVLM/BötC and RVLM/pre-BötC are pivotal to expiratory and inspiratory responses, respectively; and 2) activation of ionotropic glutamate receptors in RVLM/BötC is essential to the coupling of active expiration and sympathoexcitatory response.

Contribution of retrotrapezoid/parafacial respiratory region to the expiratory-sympathetic coupling in response to peripheral chemoreflex in rats

Central mechanisms of coupling between respiratory and sympathetic systems are essential for the entrainment between the enhanced respiratory drive and sympathoexcitation in response to hypoxia. However, the brainstem nuclei and neuronal network involved in these respiratory-sympathetic interactions remain unclear. Here, we evaluated whether the increase in expiratory activity and expiratory-modulated sympathoexcitation produced by the peripheral chemoreflex activation involves the retrotrapezoid nucleus/parafacial respiratory region (RTN/pFRG). Using decerebrated arterially perfused in situ rat preparations (60–80 g), we recorded the activities of thoracic sympathetic (tSN), phrenic (PN), and abdominal nerves (AbN) as well as the extracellular activity of RTN/pFRG expiratory neurons, and reflex responses to chemoreflex activation were evaluated before and after inactivation of the RTN/pFRG region with muscimol (1 mM). In the RTN/pFRG, we identified late-expiratory (late-E) neurons (n = 5) that were silent at resting but fired coincidently with the emergence of late-E bursts in AbN after peripheral chemoreceptor activation. Bilateral muscimol microinjections into the RTN/pFRG region (n = 6) significantly reduced basal PN frequency, mean AbN activity, and the amplitude of respiratory modulation of tSN (P < 0.05). With respect to peripheral chemoreflex responses, muscimol microinjections in the RTN/pFRG enhanced the PN inspiratory response, abolished the evoked late-E activity of AbN, but did not alter either the magnitude or pattern of the tSN reflex response. These findings indicate that the RTN/pFRG region is critically involved in the processing of the active expiratory response but not of the expiratory-modulated sympathetic response to peripheral chemoreflex activation of rat in situ preparations.