Research reportElectrophysiological characterization of facilitated spinal withdrawal reflex to repetitive electrical stimuli and its modulation by central glutamate receptor in spinal anesthetized rats
Introduction
Animal studies have shown that spinal cord dorsal horn (DH) nociceptive neuron, such as wide-dynamic range (WDR) neurons, become ‘hyperexcitable’ following repeated electrical C-fiber stimuli at low frequency (>0.3 Hz). This short-term frequency- and intensity-dependent progressive facilitation of the spinal DH neuron responses to peripheral repetitive noxious stimulation is termed as “wind-up” [15]. After first described in 1966, wind-up has been as a quite ‘famous’ artificial phenomenon of central temporal summation and has been investigated thoroughly with regard to “central sensitization” in animals and humans in the last decades [1], [2], [4], [5], [7], [11], [12], [13], [19], [28]. Nevertheless, it is important to know that nonnegligible differences excite between wind-up and central sensitization as it has been demonstrated that wind-up is the distinct phenomenon to central sensitization [31]. Central sensitization can occur without eliciting wind-up [31], but sufficient stimulation that produces wind-up in DH neurons may probably evoke central sensitization. Thus, wind-up may be only the prelude or the initial steps of the process of central sensitization. Concomitant with the wind-up phenomenon, it has also been widely observed that the WDR neurons and the single motor units (SMUs) continue to fire (termed as after-discharges) after cessation of the noxious repetitive electrical stimuli or tonic strong mechanical stimuli [2], [23], [28], [33], [34]. Compared with wind-up, the long lasting after-discharge of DH WDR neurons might be more critical for the pronounced neuronal hyperexcitability and induced central sensitization due to the possible central temporal and/or spatial summation. As a matter of fact, the after-discharges phenomenon has generally been considered to be the correlate of clinic spontaneous pathological pain such as obstinate, refractory pain of neuroma [2], [7], [23]. The possible immanent relationship among wind-up, after-discharge, and central sensitization has not, however, been studied systematically in intact animals, especially in spinalized animals at present.
With respect to the possible central pharmacological receptors involved in wind-up phenomenon, the N-methyl-d-aspartate (NMDA) receptor antagonist blocks the wind-up of the WDR neurons [4], [5], [22], [32], but interestingly it does not reduce their initial response to a series of volleys in C-fibers [4]. Findings have further suggested a potential clinical use of NMDA-antagonists such as ketamine in the management of neurogenic pain [6], [32], in particular ‘wind-up like pain’ [7], [26]. Thus, we have reason to believe that the wind-up phenomenon of DH neuron response is highly dependent on the activation of central NMDA receptor. Additionally, the non-NMDA receptor, the neurokinin-1 (NK1) receptor, and others are also proved to be involved in the wind-up process [3], [5], [13].
Price [18] showed that electromyographic (EMG) responses (flexor reflexes), recorded from hind limb muscles in the cat, could be used as an indicator for central temporal summation of the nociceptive afferent activity as repetitive electrical stimuli cause a facilitation of the reflex amplitude. Facilitation of the withdrawal reflex as an electrophysiological measure for temporal summation has been widely validated in experimental human studies [1], [2], [19]. Recently, Herrero and Cervero [12] have further developed a model where the SMU action potentials in intact and spinalized animals could also be used to quantify the withdrawal reflex and have suggested that the facilitation of spinal reflex to repetitive nociceptive stimuli is an indirect measure for the central integrative mechanism in DH neurons. Although the interest in the mechanisms and neurobiology behind wind-up of spinal DH neuron has been systematically studied, so far, the relationship between wind-up and after-discharge of spinal withdrawal reflex has not been clarified, and neither has the DH nociceptive neuron responses. In addition, detailed information regarding the role of central NMDA and non-NMDA receptor in modulation of wind-up process and after-discharge of spinal reflex is also sporadically reported.
The aims of the present study performed in spinalized animal were to systematically investigate (1) the facilitation, saturation, and/or decline of the withdrawal reflex, (2) the after-discharge following series of repetitive electrical stimuli of different intensities and frequencies, and (3) central pharmacological mechanism underlying wind-up and after-discharge using NMDA and non-NMDA antagonists.
Section snippets
Materials and methods
Male Wistar rats weighing 250–350 g were used in the present experiment. Animals were provided by the Animal Facilities of Aalborg Hospital, and housed pairwise under a 12:12 h light–dark cycle with food and water available ad libitum. The procedures were approved by the institutional Animal Ethics Committee. The Principles of Laboratory Animal Care (NIH publication No. 86-23, revised 1985) were followed, and efforts were made to minimize the suffering and reduce the number of animals used.
Results
A total of 52 SMUs from the gastrocnemius muscle were recorded in 52 Wistar male rats. Additionally, three DH WDR neurons from lumbar spinal cord were also recorded for comparative study. Only one motor unit was investigated in a 1-day experiment.
In spinal halothane-anesthetized (0.5%) rats, the transcutaneous suprathreshold, such as 1.5×T intensity of electrical stimulation applied to cRF, elicited different responses of SMUs (n=12) from gastrocnemius muscle compared with the simultaneously
Discussion
The present animal study performed in spinal anesthetized rats supports our previous human and animal studies [1], [2], [33], [34], showing that the spinally organized withdrawal reflex assessed by recording SMU EMG activity from gastrocnemius muscle can be used to evaluate the facilitation of central component-involved phenomenon, such as wind-up and after-discharge, which are markedly stimulus frequency and intensity dependent. In accordance with previous experiment in humans [1], [2], our
Acknowledgements
This study is supported by the Danish National Research Foundation (DNRF). The authors do appreciate the staff of the Animal Facilities, Aalborg Hospital, for their assistance and valuable support during the entire experiment, and Dr. Anne S. Schmidt for her help in language edition.
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