Translational value of detection and quantification of spontaneous activity and peripheral sensitization from nociceptors in animal models of neuropathic pain and in neuropathic pain patients J. Serra, E. Garcia, R. Sola, F. Antonelli, M. Sumalla, C. Gias, M. Jones, C. Quiles. MC Mutual, Barcelona, Spain; Neuroscience Technologies Ltd, London, UK. Introduction Different rodent models trying to mimic neuropathic pain in humans have been used over the last decades. However, most of these animal models rely on the detection of evoked pain, whereas the main complaint in human patients is spontaneous pain. As detection of spontaneous pain in animals is still problematic, current and recently developed behavioral methods may still have poor translational value into the clinic. Detection of abnormal spontaneous activity in nociceptors with microneurography is an attractive alternative to assess spontaneous pain. Recent findings strongly indicate that microneurography can be used to detect spontaneous activity in nociceptors as a true biomarker of spontaneous pain. Material and Methods Microneurography Raster plots of latencies of multiple, individual action potentials were recorded simultaneously from peripheral C-nociceptors using microneurography. Specifically developed software was used to sort individual action potentials, identify abnormal spontaneous activity, and quantify the number of extra spikes per unit of time. Stimulus evoked responses to mechanical and heat stimuli were also obtained. Animals and subjects Studies were usually performed using male Sprague- Dawley rats with different models of neuropathic pain: focal traumatic nerve injury (crush (n=80), suture (n=20), chronic constriction injury (n=20), tibial nerve transection n=30) and diffuse nerve pathology (streptozotocin (n=60), ddC (n=20), d4T (Wistar, n=20)), and neuropathic pain patients with focal traumatic nerve injury and small fiber neuropathies of different etiologies (n=28). Results were compared between species and between etiologies. Detection of abnormalities in C-nociceptors The following items were systematically assessed: 1. Presence of spontaneous activity in C-nociceptors 2. Presence of peripheral sensitization 3. Presence of multispikes 4. Changes in axonal membrane properties Quantification of spontaneous activity A method to quantify the intensity of spontaneous activity was developed based on an estimation of the number of extra spikes from the raster plot of latencies. Pharmacological intervention Pharmacological intervention with different agents was able to modulate or suppress such abnormal activity (e.g. with i.v. lidocaine). Results Microneurography can be performed in animals and humans using the same methods (Fig. 1). Activity-dependent slowing of conduction velocity allows segregation of C-fibers into different functional groups (Figure 2). Abnormal spontaneous activity was regularly seen both in animal models (Fig. 3) and patients (Fig. 4), almost exclusively in mechano-insensitive C-nociceptors. Comparison of different measures of spontaneous activity showed no differences between different rat models and neuropathic pain patients (Fig. 5). The proportion of abnormally spontaneous C-nociceptors varies between models, being higher in focal nerve injury, and lower in polyneuropathy states (Fig. 6). Other abnormal findings were also regularly seen: peripheral sensitization to mechanical and heat stimuli, double and triple spikes, and “peripheral” wind up-like phenomena (Fig. 7). Pharmacological intervention is possible, with direct translational value between animals and humans (Fig. 8). SS7: Study design, analysis and experimental reporting. Official Satellite Symposium of the 14th World Congress on Pain, IASP, Milan 2012 Figure 2: Modified raster plot showing latency profiles of normal, quiescent C-fibers. Note the different patterns of latency slowing during a period of 2Hz stimulation, allowing the identification of nociceptors (slowing >15% of baseline latency). The trace also illustrates different patterns of recovery to baseline latency following a return to 0.25 Hz stimulation. The recording was made in a sham-operated rat. Figure 3: Modified raster plot showing abnormal ‘saw-tooth’ profile of latency in two mechano-insensitive C-nociceptors recorded in an animal with a sciatic nerve crush injury. Each of the ’saw teeth’ represents a slowing of the latency of evoked spikes due to the generation of spontaneous spikes in the interval between electrical stimuli. Figure 4: Modified raster plot showing abnormal ‘saw-tooth’ profile of latency in a mechano-insensitive C-nociceptor recorded in a patient with fibromyalgia. As in Figure 3, each of the ’saw teeth’ represents slowing of the latency of evoked spikes due to spontaneous extra spikes firing. Figure 6: Changes in spontaneous activity in different rat models with time. The proportion of spontaneously active mechano-insensitive C- nociceptors, grouped by number of days since insult (in the case of ddC, days since last injection). Note different time scales for the different rat models. Conclusion Detection and quantification of spontaneous activity in identified subpopulations of peripheral C-nociceptors is a powerful method to test the efficacy of new compounds targeting peripheral nociceptor hyperexcitability. The method can be used both in animals and in humans. Acknowledgements: some of this work was part of the IMI Europain project and funded by the IMI JU, grant 11507. Figure 1: Microneurography was originally developed to record individual action potentials from peripheral nerve axons in humans. It is a minimally invasive technique that has been performed on thousands of patients, without significant side effects. It has recently been adapted to the animal setting, and there is considerable experience with microneurographic recordings in rat. A conspicuous finding in neuropathic pain patients and in animal models of neuropathic pain is the presence of abnormal spontaneous activity in C-nociceptors. 0.25 Hz 0.25 Hz 0.25 Hz 2 Hz 0 Hz 0.25 Hz 0.25 Hz 0.25 Hz 2 Hz 0 Hz Figure 5: Examples of spontaneous discharges in mechano-insensitive C-nociceptors in five different rat models of neuropathic pain (from top to bottom, left to right): crush (55 days), suture (56 days), CCI (15 days), STZ (56 days), and ddC (14 days after last injection). Bottom right: An unmodified raster plot from a painful diabetic neuropathy patient also displays multiple C- nociceptor units with irregular saw-tooth baselines indicative of spontaneous activity. Figure 7: Other abnormal findings were also regularly seen both in animals and in neuropathic pain patients. Top left: peripheral sensitization to mechanical (M) and heat (H) stimuli in normally insensitive “silent” C-nociceptors (arrows correspond to periods of mechanical and heat stimulation). The unit, which is engaged in spontaneous activity, vigorously responds to these stimuli. Top right: “peripheral” wind up-like phenomena. Repetitive stimulation of the unit with mechanical stimuli (arrows) gives rise to progressively larger responses that may last for several minutes. Bottom: stimulation with single electrical pulses to the receptive field of C-fibers may occasionally produce two or even three spikes, instead of the normal one, due to unidirectional block of branching points of the terminal arborization of nociceptors in the skin. ISI between action potentials are closely spaced, inducing clearly additive EPSP on spinal cord second order nociceptive neurons. A: single sweep at the time pointed by arrow in raster plot B. Three C-fibers are multispiking. At shorter latency a C-cold thermoreceptor gives a double spike (a,b), and at longer latencies two nociceptors give double (c,d) and triple spikes (e,f,g). Figure 8: Examples of the effect of a test compound on spontaneous activity in a pathological C-fiber (left: confidential compound, right: i.v. lidocaine 5 mg/kg). Both recordings obtained from rats with a sciatic nerve crush injury. Latency raster plots illustrating an irregular “saw tooth” baseline latency due to bursts of spontaneous activity in mechano-insensitive C- nociceptors. The arrows indicate ongoing bursts of spontaneous impulses. Left: A confidential test compound was administered systemically at minute 50. Approximately 15 min later, the latency baseline became stable, indicating that the nociceptor unit had stopped its spontaneous discharge. Right: i.v. lidocaine was infused systemically from minute 90 to 100, with no clear modulation of ongoing spontaneous activity. M M M M H H H View publication stats View publication stats