Carsten Dahl Mørch

Simultaneous recordings of wind-up of paired spinal dorsal horn nociceptive neuron and nociceptive flexion reflex in rats

To study the sensory-motor interaction of spinal processing underlying the neuronal mechanisms of the nociceptive flexion reflex (NFR) and its temporal facilitation, 16 spinal dorsal horn (DH) wide-dynamic-range (WDR) neurons and paired 16 single motor units (SMU) from the gastrocnemius soleus muscle (GS) were simultaneously recorded using extracellular single unit and electromyographic techniques in spinal, halothane-anesthetized rats. The paired DH WDR neuron and GS SMU showed a parallel increase in the firing rate and duration of spike responses to noxious pinch stimuli applied to their common cutaneous receptive field (cRF) on the ipsilateral hind paw skin. Innocuous brush or pressure evoked no, or less, firing in the SMU but evoked a graded increase in spike responses in the simultaneously-recorded WDR neuron. Moreover, both pressure and noxious pinch stimuli evoked a short-lasting after-discharge (for several min) in the WDR neuron but without any after-discharge in the simultaneously-recorded SMU. The paired WDR neuron and SMU also showed a parallel basal response (termed as early and late components according to latency), after-discharge and wind-up of the late response to repetitively applied supra-threshold electrical stimulation (intensity: >1.5 T, duration: 1 ms and frequency: 1 Hz for 15 s). Linear regression and cross-correlation histogram analyses showed that the DH WDR neuron had a significant correlation with the simultaneously-recorded SMU and they were functionally located in the spinally-organized NFR circuitry via polysynaptic connections. Systemic administration of fentanyl, an opioid receptor agonist, resulted in a parallel, naloxone-reversible suppression of both basal late response component and wind-up response in both WDR neuron and SMU paired; however, fentanyl suppressed only the early response of the SMU without any effect on that of the DH WDR neuron. The present results provide new direct evidence showing an essential role of spinal DH WDR neurons in the mediation of spinally-organized NFR as well as its temporal facilitation (wind-up). Based on these data, the spinal DH WDR neuron seems to function as a signal discriminator or frequency encoder of multireceptive primary afferent impulses that may determine excitable level of motor output and the occurrence of a behavioral NFR via polysynaptic connections. Consequently, the spinal WDR neuron-mediated NFR and its temporal facilitation are likely to be modulated by spinal endogenous opioid peptides via opioid receptors on the nociceptive sensory components of the spinally-organized NFR circuitry.

Convergence of cutaneous, musculoskeletal, dural and visceral afferents onto nociceptive neurons in the first cervical dorsal horn

The convergence of cutaneous, musculoskeletal, dural and visceral afferents onto nociceptive neurons in the first cervical dorsal horn was investigated in urethane/chloralose‐anesthetized rats. Electrical stimulation was applied to facial, neck, shoulder and forepaw skin, cornea (COR), dura, second cervical (C2) nerve, hypoglossal nerve, temporomandibular joint, masseter (MAS) muscle and superior laryngeal nerve. In addition, acetic acid was injected intraperitoneally and microinjection of glutamate was applied to the tongue, MAS muscle, splenius cervicis muscle, dura and intrapericardial area. A total of 52 nociceptive neurons classified as wide dynamic range (n = 28) or nociceptive‐specific (n = 24) was studied. All nociceptive neurons received afferent input from the skin and at least one COR, musculoskeletal, dural or visceral afferent source in the trigeminal (V) or cervical area but input from afferent sources caudal to the C2 innervation territory was sparse. The proportion of neurons responding to COR, dural, C2 nerve, hypoglossal nerve, temporomandibular joint, MAS muscle and superior laryngeal nerve stimulations was 87, 54, 85, 52, 73, 64 and 31%, respectively. Electrical stimulation of all tested sites showed a double logarithmic stimulus–response relation, and cluster analysis of the excitability to COR, musculoskeletal, dural and visceral stimulations revealed two groups of neurons, one mainly containing wide dynamic range neurons and one mainly containing nociceptive‐specific neurons. These findings indicate that afferent convergence in first cervical dorsal horn nociceptive neurons may be limited to the craniofacial area and that they may play an important role in the integration of craniofacial and upper cervical nociceptive inputs.

Differential effect of peripheral glutamate (NMDA, non-NMDA) receptor antagonists on bee venom-induced spontaneous nociception and sensitization.

This study aimed to investigate the role of peripheral N-methyl-d-aspartate (NMDA) and non-NMDA receptor on (1). spontaneous nociception and (2). on sensitization induced by subcutaneous (s.c.) injection of bee venom (0.2mg/50 micro l) in rats. Peripheral s.c. administration of the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP5), the non-competitive NMDA receptor channel blocker MK-801, and the competitive non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were performed before (pre-treatment) and after (post-treatment) bee venom-induced inflammation. Pre-treatment with AP5 (10mM, 50 micro l) and both pre-treatment and post-treatment with MK-801 (2mM, 50 micro l) into the same area of the bee venom injection site markedly reduced the bee venom-increased spontaneous responses of wide-dynamic range (WDR) neuron of the spinal cord. Post-treatment with the same dose of AP5 as well as pre-treatment and post-treatment with CNQX (5mM, 50 micro l) did not produce any inhibitory effects. Additionally, the role of peripheral NMDA and non-NMDA receptors on bee venom-induced mechanical allodynia and hyperalgesia were investigated and assessed by the paw withdrawal reflex to the innocuous and noxious mechanical stimulation. Peripheral administration of AP5, but not CNQX, reduced mechanical allodynia and hyperalgesia. The data suggest that the peripheral NMDA receptor, but not non-NMDA receptor, plays a pivotal role in the bee venom-induced persistent nociception and hyperexcitability.

Is the conditioned pain modulation paradigm reliable?: A test-retest assessment using the nociceptive withdrawal reflex

The aim of this study was to determine the reliability of the conditioned pain modulation (CPM) paradigm assessed by an objective electrophysiological method, the nociceptive withdrawal reflex (NWR), and psychophysical measures, using hypothetical sample sizes for future studies as analytical goals. Thirty-four healthy volunteers participated in two identical experimental sessions, separated by 1 to 3 weeks. In each session, the cold pressor test (CPT) was used to induce CPM, and the NWR thresholds, electrical pain detection thresholds and pain intensity ratings after suprathreshold electrical stimulation were assessed before and during CPT. CPM was consistently detected by all methods, and the electrophysiological measures did not introduce additional variation to the assessment. In particular, 99% of the trials resulted in higher NWR thresholds during CPT, with an average increase of 3.4 mA (p<0.001). Similarly, 96% of the trials resulted in higher electrical pain detection thresholds during CPT, with an average increase of 2.2 mA (p<0.001). Pain intensity ratings after suprathreshold electrical stimulation were reduced during CPT in 84% of the trials, displaying an average decrease of 1.5 points in a numeric rating scale (p<0.001). Under these experimental conditions, CPM reliability was acceptable for all assessment methods in terms of sample sizes for potential experiments. The presented results are encouraging with regards to the use of the CPM as an assessment tool in experimental and clinical pain. Trial Registration: Clinical Trials.gov NCT01636440

Effects of adipose thickness and muscle hardness on pressure pain sensitivity

ObjectivePressure algometry is used for assessment of pain sensitivity. In this study the relation between tissue characteristics and pressure pain thresholds was investigated. MethodsThree-dimensional finite-element computer-models were developed to simulate the tissue stress and strain distribution during pressure stimulation on muscles with different hardness (I, II, III, IV (hardest)) and subcutaneous adipose tissue thickness (normal and thicker). The computer model was validated based on data recorded by computer-controlled pressure-induced muscle pain in 8 and 16 partecipants, respectively. ResultsThe experimental pressure-indentation curve fitted the outcome of the FE model (R2>0.73). Stress and strain were extracted from the models at a known painful pressure stimulation level. PPT and PPTO were not significantly different in subjects with normal and thick adipose tissue in accordance with the simulation model where the strain in muscle tissue was comparable in the two conditions. The strain in adipose tissue was larger in subjects with thick adipose tissue compared with normal adipose thickness. In relaxed muscle (hardness I) the principal strain peaked at 0.12 in the adipose tissue, was reduced to 0.07 in the muscle tissue and 0.05 in the harder muscle. Significantly higher PPT and PPTO were recorded in harder compared with softer muscles (P<0.02). DiscussionThe pressure pain sensitivity of the deep layer is related to the amount of muscle strain, which is affected by the muscle hardness and the thickness of adipose tissue. This is clinically relevant as these two factors are not taken into consideration when pressure pain assessments are performed in clinical routine.

Electrophysiological characterization of facilitated spinal withdrawal reflex to repetitive electrical stimuli and its modulation by central glutamate receptor in spinal anesthetized rats.

The present study is aimed to systematically investigate wind-up and after-discharge of the spinal withdrawal reflex assessed by recording single motor unit (SMU) electromyographic (EMG) response to different intensities [0.5-1.5xreflex threshold (T)] of repetitive [frequencies (0.5-200 Hz)] transcutaneous electrical stimuli for 5 s. The role of central glutamate receptors in modulation of the withdrawal reflex facilitation was observed and evaluated in order to explore the potential central mechanism. Stimulus intensities below reflex threshold, such as 0.8xT, but not 0.5xT, could by repetition elicit and facilitate withdrawal reflex. The facilitation (wind-up and after-discharge) of the withdrawal reflex is a result of central integration and is increased significantly for increasing stimulus intensity and frequency. Electrical stimuli at 3-5 Hz for 5 s are appropriate to elicit wind-up. In contrast, 10-20 Hz frequencies of electrical stimuli are adequate to evoke the after-discharge. For pharmacological intervention, suprathreshold (1.5xT) repeated (5 Hz) electrically evoked facilitated reflex (wind-up) were apparently depressed by intrathecal (i.t.) administration of MK-801 as well as CNQX (40 nmol/10 microl, respectively). However, wind-up of spinal reflexes evoked by subthreshold (0.8xT) electrical stimuli could only be depressed by the treatment with CNQX, not MK-801. The after-discharge of the withdrawal reflex elicited by 20 Hz electrical stimulation with either 0.8xT or 1.5xT intensity was depressed by i.t. treatment with CNQX. I.t. application of MK-801 only depressed 0.8xT the intensity of electrically evoked after-discharge. In conclusion, for the first time, the present study clearly demonstrates that, following the wind-up phase, the spinal withdrawal reflex pathways continue to fire spontaneously in a stimulus frequency- and intensity-dependent way (temporal and/or spatial summation). This inherited memory and the central non-N-methyl-d-aspartate (non-NMDA) receptor, but not the NMDA receptor, mainly involving pharmacological mechanisms, may play an important role in pathological conditions with spontaneous nociceptive firing. Furthermore, the after-discharge of the spinal reflex may be an important indicator for studies on central sensitization in many pathological pain conditions.

Exteroceptive aspects of nociception: insights from graphesthesia and two-point discrimination.

&NA; The exteroceptive capabilities of the nociceptive system have long been thought to be considerably more limited than those of the tactile system. However, most investigations of spatio‐temporal aspects of the nociceptive system have largely focused on intensity coding as consequence of spatial or temporal summation. Graphesthesia, the identification of numbers “written” on the skin, and assessment of the two‐point discrimination thresholds were used to compare the exteroceptive capabilities of the tactile and nociceptive systems. Numbers were “written” on the forearm and the abdomen by tactile stimulation and by painful non‐contact infrared laser heat stimulation. Subjects performed both graphesthesia tasks better than chance. The tactile graphesthesia tasks were performed with 89% (82–97%) correct responses on the forearm and 86% (79–94%) correct responses on the abdomen. Tactile graphesthesia tasks were significantly better than painful heat graphesthesia tasks that were performed with 31% (23–40%) and 44% (37–51%) correct responses on the forearm and abdomen, respectively. These findings demonstrate that the central nervous system is capable of assembling complex spatio‐temporal patterns of nociceptive information from the body surface into unified mental objects with sufficient accuracy to enable behavioral discrimination.

Differential antinociceptive effects induced by a selective cyclooxygenase-2 inhibitor (SC-236) on dorsal horn neurons and spinal withdrawal reflexes in anesthetized spinal rats.

The aim of present study was to examine the effect of a selective cyclooxygenase-2 (COX-2) inhibitor SC-236 (4 mg/kg) on the simultaneous responsiveness of spinal wide-dynamic range (WDR) neurons and single motor units (SMUs) from gastrocnemius soleus muscles to mechanical stimuli (pressure and pinch) and repeated suprathreshold (1.5xT, the intensity threshold) electrical stimuli with different frequencies (3 Hz, 20 Hz) under normal conditions and bee venom (BV, 0.2 mg/50 microl)-induced inflammation and central sensitization. During normal conditions, the responses of SMUs, but not WDR neurons, to mechanical and repeated electrical stimuli (3 Hz, wind-up) were depressed by systemic administration of SC-236 as well as its vehicle (100% dimethyl sulfoxide (DMSO)). The after-discharges of both the WDR neurons and the simultaneously recorded SMUs after electrical stimuli with 20 Hz were markedly depressed only by SC-236, indicating that the mechanisms underlying the generation of the C-fiber mediated late responses and the after-discharges may be different. The enhanced responsiveness of both WDR neurons and SMUs to mechanical pressure stimuli (allodynia) and pinch stimuli (hyperalgesia) in the BV experiments was apparently depressed by SC-236, but not its vehicle. For electrical stimulation, the enhanced late responses and after-discharges, but not early responses, of both the WDR neurons and the simultaneously recorded SMUs were markedly depressed only by SC-236. This indicates that different central pharmacological mechanisms underlie the generation of these enhanced early, late responses, and after-discharges during BV-induced inflammation. The data suggest that the COX-2 inhibitor SC-236 apparently depress the activities of both spinal cord dorsal horn neuron and spinal withdrawal reflex during BV-induced sensitization, indicating that COX-2 plays an important role in the maintenance of central sensitization.

Role of central NMDA versus non-NMDA receptor in spinal withdrawal reflex in spinal anesthetized rats under normal and hyperexcitable conditions

The present study aimed to investigate the role of central N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the spinal withdrawal reflex assessed by recording single motor unit (SMU) electromyogram (EMG) response to peripheral mechanical (pressure, pinch) stimuli and repeated electrical stimuli at 3 and 20 Hz. During normal conditions, intrathecal administration of MK-801 and CNQX apparently depressed mechanically and electrically (3 Hz) evoked EMG responses in a dose-dependent manner (10, 20 and 40 nmol in 10 microl). In contrast, the after-discharges to 20 Hz electrical stimuli were suppressed only by CNQX treatment, not by MK-801 treatment. This indicates that the central mechanisms underlying the different frequencies of electrically evoked withdrawal reflex may be different. During peripheral bee venom (BV, 0.2 mg/50 microl) induced inflammation and central sensitization, the enhanced SMU EMG responses including after-discharges to pinch stimuli and 3 Hz electrical stimuli were depressed significantly by treatments with both MK-801 and CNQX. However, the enhanced SMU activities to innocuous pressure stimuli were depressed only by treatment with CNQX. Likewise, enhanced long lasting after-discharges elicited by 20 Hz electrical stimuli were also only depressed by CNQX, indicating that different central mechanisms are involved in the persistent hyperexcitability during BV-induced inflammation. The data suggest that both central NMDA and non-NMDA receptors play important roles in the transmission of nociceptive information under normal conditions. In BV-induced inflammation, however, central non-NMDA receptors, but not NMDA receptors, play a pivotal role in the generation of persistent hyperexcitability to mechanical and electrical stimuli at different frequencies (3 Hz, 20 Hz).

Long-term facilitation of nociceptive withdrawal reflexes following low-frequency conditioning electrical stimulation: A new model for central sensitization in humans

Central sensitization is believed to be one of the key mechanisms behind chronic pain conditions, and several models have been developed in order to characterize this phenomenon in humans. One of these models relies on conditioning electrical stimulation to elicit long‐lasting effects on the nociceptive system. The aim of this study was to evaluate these effects using an objective electrophysiological measurement, the nociceptive withdrawal reflex (NWR). Long‐term changes in spinal nociception after high‐ and low‐frequency conditioning electrical stimulation were assessed in 13 healthy volunteers. Perceptual intensity ratings to mechanical stimuli and blood flow variations were assessed in the conditioned area (dorsum of the foot) and surroundings. To evaluate the excitability of the nociceptive system, the NWR was elicited within the same innervation area (superficial peroneal nerve) at graded stimulation intensities and recorded in the hamstrings. Following low‐frequency stimulation, an intensity‐independent long‐lasting facilitation of the NWR was observed, with a significant increase in the reflex size (average of 31 ± 4%, p < 0.001) and in the number of reflexes (average increase of 22 ± 10%, p < 0.01), accompanied by a significant increase in the blood flow (average increase of 40 ± 10%, p < 0.001). These findings suggest that activity‐dependent central sensitization can be elicited using conditioning electrical stimulation with a stimulation frequency that lies within the physiological firing range of primary afferents, and that it can be objectively assessed in humans using the NWR.