Patients with peripheral neuropathic pain usually present with spontaneous ongoing pain and sometimes with additional abnormal stimulus-evoked pain (e.g., allodynia) . Allodynia is defined as “pain due to a stimulus which does not normally provoke pain” . Two types of mechanical allodynia have been distinguished in studies of neuropathic pain patients; dynamic mechanical allodynia (DMA) and static mechanical allodynia (SMA) [3,4]. Clinically, DMA is evoked by a normally innocuous light moving mechanical stimulus on the skin and SMA by a sustained, normally innocuous pressure against the skin. In neuropathic pain patients, with the exception of, e.g., subgroups of patients with post-herpetic neuralgia and nociceptor sensitisation  DMA is claimed to be mediated by myelinated fibres [3,4,6,7] and SMA by C-fibres [3,4,6] but also A-delta fibres have been implicated in the static subtype [7,8].
Moving a brush across the non-inflamed skin surface is likely to result in dynamic activation of both rapidly adapting (RA) and slowly adapting (SA) mechanoreceptors [9,10,11]. A brief vertical, about 1 s, stimulation of the skin with a thin von Frey filament causes a short deformation of the skin surface and an “on-off” activation of A-beta mechanoreceptors . Hence, a vertical deformation of the skin induced by stimulation with a von Frey filament activates the same peripheral substrate as a horizontally moving brush. Under normal conditions a light sustained non-painful pressure applied to the skin also results in activation of A-beta mechanoreceptors . In patients with peripheral neuropathic pain and SMA, based on the aforementioned published data, a sustained, about 10 s, indentation of the skin with a von Frey filament would hypothetically result in activation of epidermal C-and A-delta nociceptors [14,15].
With these physiological and pathophysiological concepts, partly with experimental support from the literature and partly hypothetical, we aimed at investigating if short (1 s) or longer (10 s) lasting usually non-painful von Frey filament stimulation of the neuropathic skin could be used to assess perception thresholds to DMA and SMA. Techniques to quantify the different allodynias at perception threshold level are in demand as adjuncts to suprathreshold stimuli in intervention studies aimed at modifying these stimulus-evoked phenomena 
Eighteen patients with painful unilateral partial peripheral traumatic nerve injury in a limb were studied. Patients were all outpatients recruited from Pain Center, Dept. of Neurosurgery, Karolinska University Hospital, Solna and were diagnosed with neuropathic pain by a neurologist, i.e., author P.H. or Å.L. Since all patients had an ongoing or previous contact with Pain Center they could be identified through patient charts. If the inclusion criteria were fulfilled the patient was contacted by author Å.L. and thoroughly interviewed with the purpose not to include patients meeting any exclusion criteria. No patient had any previous experience with a compression/ischemia-induced (differential) nerve block. Inclusion criteria were duration of pain >6 months and only patients with DMA with or without concomitant SMA were eligible. Exclusion criteria were complete nerve lesions, bilateral nerve lesions, clinical signs of overt neurogenic inflammation or autonomic dysfunction, a diagnosis of CRPS type II, age <18 or >80 years, uncontrolled hypertension, pain of non-neuropathic origin in the affected or contralateral area, systemic diseases predisposing for neuropathy or severe somatic or psychiatric diseases. If the patient was treated with a spinal cord stimulator this had to be turned off for at least 12 h before examination to allow for the pain relieving effect to cease. Ongoing pharmacological treatment of the painful condition was allowed.
DMA was considered to be present if pain was evoked by stroking the neuropathic skin with a camel’s hair brush, and SMA if sustained prodding perpendicularly against the skin for 10 s using a q-tip was painful for the duration of the stimulation. The study was performed in accordance with the Declaration of Helsinki and was approved by the local ethical committee of the Karolinska University Hospital, Solna. All patients gave their informed consent to participation.
2.2 General procedure
The diagnosis of peripheral neuropathic pain was based on a history of nerve injury, a neuroanatomically correlated distribution of pain in conjunction with findings of sensory abnormalities within the area of pain at bedside examination. To guide sensibility testing the patients were asked to indicate the area of spontaneous ongoing pain and DMA/SMA on a body drawing. All patients underwent a neurological examination including a bedside examination of somatosensory systems (touch, warmth, cold and pin prick) as part of the diagnostic work-up. All tests and sensibility assessments were performed by author Å.L. in a quiet room with the patient comfortably seated in a chair or lying in a relaxed supine position on a bed. Before the test session patients were asked to rate the intensity of spontaneous ongoing pain on a 100 mm visual analogue scale (VAS). The skin area with DMA/SMA was identified and outlined with a pen. To assure that the subsequent assessments using von Frey filament and thermal testing were made in the same location an examination area was marked with a pen within the area of DMA. In patients with concomitant SMA the position of the examination area was located where both DMA and SMA could be evoked. Special care was taken to apply the thermode within the area of DMA where the entire surface of the thermode was in contact with the allodynic skin. Before start of the test session the patients were carefully familiarized with the different methods to be used and to the testing procedure. The patients were instructed to keep their eyes closed during the tests and were unaware of the test results during the session. Before start of the nerve block baseline sensibility testing, i.e., perception thresholds to light touch, cold and warmth was performed, as previously described, first in the corresponding contralateral non-injured area and then in the innervation territory of the injured nerve to get an estimation of the degree of neuropathy in the area of DMA/SMA . In the neuropathic area also pain perception thresholds to von Frey filament stimulation of 1 s and 10 s were assessed (see below). Finally, a control area with no signs of neuropathy neighbouring the neuropathic area at the same proximo-distal level was examined to obtain baseline values for cold and warm perception thresholds subsequently used (se below) to monitor the progression of a compression/ischemia-induced (differential) nerve block of the painful limb. The method of limits was used in all quantitative testing of somatosensory perception thresholds .
2.3 Quantitative sensory testing
2.3.1 Pain perception thresholds to von Frey filament stimulation
Pain perception thresholds to mechanical stimulation were assessed using a set of 15 von Frey filaments (OptiHair®, Marstock-nervtest, graded from 0.29 mN (0.03 g) to 294 mN (30 g) (logarithmical increase)) made of optical glass fibre . To keep the contact surface approximately constant for various fibre diameters the tip of the fibre is coated with a tiny round epoxy bead (diameter about 0.5 mm). This may also reduce the risk of nociceptor activation compared to conventional nylon monofilaments with sharp edges [19,20]. Care was taken to apply the filaments perpendicularly to the surface of the skin avoiding contact with body hair, shaving the skin if necessary. We used stimulus duration of approximately 1 s or 10 s aiming at assessing the perception threshold counterpart to DMA and SMA. The baseline von Frey pain perception thresholds were defined as the lowest pressure considered painful for the different stimulus duration of 1 s and 10 s, respectively. All patients were examined with both stimulus durations, the short stimulus always preceding the longer one. The average of three ascending perception levels was calculated as the baseline von Frey pain perception threshold.
Importantly, the von Frey filaments used in the study did not evoke pain in the contralateral pain free area or in the control area neighbouring the neuropathic skin in any patient.
2.3.2 Thermal perception thresholds
Thermal perception thresholds were assessed using a Peltier element based thermode of 12.5 cm2 (Modular Sensory Analyser, Somedic Sales AB, Hörby, Sweden) applied to the skin. If necessary to keep the entire thermode surface in contact with the skin the device was secured with an elastic bandage, care taken to apply minimal pressure. The baseline temperature of the thermode was set equal to the skin temperature assessed with the infrared skin temperature analyzer Tempett® (Somedic Sales AB, Hörby, Sweden) and then, if necessary adjusted manually until the patient perceived the sensation of the thermode as indifferent. The baseline perception thresholds to non-painful cold and warmth were obtained by delivering five cold followed by five warm stimuli with a preset randomised inter-stimulus interval of 4–10 s and with a stimulus rate of 1 °C/s. The patients were instructed to press a hand-held button at the first sensation of cold or warmth, respectively, thereby terminating the stimulus. Thresholds were calculated as the average temperature difference from skin temperature of the five successive perception levels. To avoid tissue damage the maximum and minimum temperatures were set at 50 and 5 °C, respectively. Failure to respond before the cut-off limit was reached resulted in assignment of the cut-off value.
2.4 Compression-ischemia induced (differential) nerve block
To study which nerve fibre population that contributes to pain at 1 and 10 s prodding of the skin with von Frey filaments a compression/ischemia-induced (differential) nerve block approach was used [4,21]. This was obtained through inflation of a sphygmomanometer cuff proximally placed around the symptomatic limb, inflating it to a level of 80–100 mm Hg above the systolic blood pressure . Shortly after inflating the cuff all patients experienced spontaneous non-painful paresthesias in the painful limb. This sensation vanished after a few minutes. The patients were carefully instructed not to move the limb during the course of the block since this may induce paresthesias or pain which may affect the outcome of the testing procedure.
During the nerve block single perception levels to cold (CL) and warmth (WL) were assessed every 1–3 min in the control area. This was done to monitor function in A-delta (cold) and C-fibres (warmth) during progression of the block. A significant elevation of a thermal perception level during the nerve block was defined as a sustained increase of at least 2 standard deviations (SD) compared to the individual pre-block mean. During the block also single von Frey pain perception levels to 1 s (vF1) and 10 s (vF10) stimulation were repeatedly assessed every 1–3 min in the neuropathic area by single ascending stimuli. An increase in the pain perception level to von Frey filament stimulation of at least 2 steps (logarithmical increase) of the bending threshold during the block compared to pre-block values was regarded a significant increase. Also, the perception magnitude from brushing (normal, increased, decreased or none) the skin with a camel’s hair brush in the control area compared to the contralateral pain free area were assessed at the same intervals to monitor A-beta-fibre function (touch). The nerve block was terminated if the patient could not tolerate the pain caused by the cuff, if the spontaneous ongoing or stimulus-evoked pain in the limb became unbearable, if a total loss of touchand cold sensation indicating block of all A-fibres was obtained or at a maximum blocking time of 45 min. If the nerve block was terminated before significant elevation of CL and WL was obtained the time point of termination was assigned as the time point for elevation of CL and WL to allow for group level statistical analysis. The time line of the experimental procedure is presented in Fig. 1. An illustrative case is depicted in Fig. 2.
Data was normally distributed. Results were analysed using a one-way ANOVA with repeated measures on factor “time” with 3 or 4 levels (time to elevation of vF1 and/or vF10, CL and WL). If the F-ratio for the main effect of “time” was significant, Fisher’s LSD test or Tukey test was performed depending on the number of factor levels. When comparing patients with and without SMA data was analysed using a two-way ANOVA with repeated measures with the between-group factor “SMA” with 2 levels (yes/no) and the within-group factor “time” with 3 levels (time to elevation of vF1, CL and WL). If a significant interaction between “SMA” and “time” was found, simple main effects tests were performed, i.e., effects of one factor holding the level of the other factor fixed. If no significant interaction with factor “time” was present and the F-ratio for the main effect of “time” was significant, Fisher’s LSD test was performed. The sphericity assumption was met in all the ANOVA models. T-test was used comparing time to elevation of vF10 in patients with SMA with time to elevation of vF1 in patients without SMA. Statistical significance was accepted at p ≤ 0.05. Data were analysed using Statistica 8.0, StatSoft®, Inc., Tulsa, OK, USA.
Table 1 presents demographic data of the 18 patients included in the study (12 females, 6 males, median age 51 years, range 24–62 years). The median duration of time since nerve injury was 5 years (range 1–12 years). Nine patients reported concomitant SMA in the area of DMA. Six patients had ongoing pharmacological treatment of their neuropathic pain. Four patients were treated with a spinal cord stimulator.
3.2 Pain perception threshold to von Frey filament stimulation
In the neuropathic area all patients reported pain from the 1 s von Frey stimulation (Table 2). Only patients with clinically established SMA (n = 9) reported sustained pain during the von Frey filament stimulation of 10 s (Table 2). Patients with only DMA reported pain during the initial 1–3 s of the total stimulus duration of 10 s and for a few s after the filament was removed.
3.3 Time to threshold elevation of vF1
In the one-way ANOVA, there was a significant difference between time to elevation of vF1, CL and WL (n = 18) during the differential nerve block (F(2, 34) = 37.65, p < 0.001). In the post hoc analysis using Fisher’s LSD test, elevation of vF1 occurred significantly prior to elevation of both CL (p < 0.001) and WL (p < 0.001) (Fig. 3).
Comparing patients with (n = 9) and without SMA (n = 9) in the two-way ANOVA, no significant main effect of group could be demonstrated (F(1, 16) = 0.02, p = 0.89). There was a significant difference between time to elevation of vF1, CL and WL during the differential nerve block within each group (F(2, 32) = 36.02, p < 0.001). This analyses did not reveal a significant interaction (F(2, 32) = 0.26, p = 0.77) between the factors “time” (time to elevation of vF1, CL and WL) and “group” (SMA yes/no) and thus no significant difference in time to elevation of vF1 between patients with 3.4. Time to threshold elevation of vF10 In the one-way ANOVA, there was a significant difference between time to elevation of vF1, vF10, CL and WL in patients with SMA (n = 9) during the differential nerve block (F(3, 24) = 17.32, p < 0.001). In the post hoc analysis using Tukey test elevation of vF10 occurred significantly prior to both elevation of CL (p < 0.05) and WL (p < 0.001) (Fig. 5).
At the time of vF1 elevation 3/18 patients (no. 1, 3, 7) presented with an elevated CL of ≥2 SD (an increase of 3.5 °C, 1.8 °C and 1.3 °C, respectively). None of the patients reported altered perception level to warmth at the time of elevation of vF1 (Table 2).
3.4 Time to threshold elevation of vF10
In the one-way ANOVA, there was a significant difference between time to elevation of vF1, vF10, CL and WL in patients with SMA (n = 9) during the differential nerve block (F(3, 24) = 17.32, p < 0.001). In the post hoc analysis using Tukey test elevation of vF10 occurred significantly prior to both elevation of CL (p < 0.05) and WL (p < 0.001) (Fig. 5).
At the time of elevation of vF10, 2/9 patients (nos. 3, 7) presented with an elevated cold perception level of ≥2 SD (an increase of 3.4 °C and 1.3 °C, respectively). None of the patients reported altered perception level to warmth at the time of elevation of vF10 (Table 2).
3.5 The relationship between vF1 and vF10
Pain due to a usually non-painful mechanical stimulus, mechanical allodynia, is an oppressive symptom in subgroups of patients with neuropathic pain interfering extensively with the patients activity of daily living in addition to spontaneous ongoing pain. Techniques to assess different allodynias at perception threshold level are in demand as adjuncts to suprathreshold stimuli in intervention studies aimed at modifying these stimulus-evoked phenomena . Pain induced by usually non-painful von Frey filament prodding of the skin has been reported on in patients with neuropathic pain and may be a useful approach if the type of stimulation could be linked to activation of specific peripheral nerve fibres .
The main outcome of this study was the finding that elevation of both vF1 in patients with DMA and vF10 in patients with SMA occurred concurrently in time and significantly prior to an increase in the perception level to cold during the continuous nerve block, pointing to the involvement of A-beta fibres as the peripheral substrate. Single patients demonstrated a slight decrease in cold perception levels at the time of elevation of vF1 or vF10 and a possible contribution to mechanical allodynia from A-delta-fibres can therefore not completely be ruled out although the recorded alterations were minor. None of the patients reported an elevation of the perception level to warmth at the time of elevation of vF1 or vF10 excluding contribution from C-fibres. It is widely accepted that during a compression/ischemia induced nerve block conduction in myelinated fibres is blocked successively depending on thickness and starting at an early phase and prior to unmyelinated fibres [21,23,24]. It has been claimed, although not observed in the present study, that the sequence of blocking within the myelinated fibre group is insufficiently differentiated by such an approach as shown by the nearly simultaneous disappearance of the sensation of light touch (A-beta) and cold (A-delta) [25,26].
Further, in the present study only patients with clinically established SMA (n = 9) reported continuous pain to a sustained 10 s von Frey filament stimulation (vF10). Patients with only DMA (n = 9) reported pain merely for the initial 1–3 s of the total stimulus duration of 10 s and for a few seconds after the filament was lifted from the skin. In the study by Ochoa and Yarnitsky SMA in patients with neuropathic pain persisted in a majority (15/18) of patients during a compression/ischemia nerve block although diminished in intensity (in 10/15 patients) when loss of cold and touch sensation was established and warm sensation remained unaltered . The result was interpreted by the authors as an indication that SMA predominantly was mediated by C-fibres .
Regarding the possible involvement of A-delta fibres in mediating SMA in the present study we monitored cold-activated A-delta fibres during the block but did not explicitly test the function of A-fibre nociceptors, i.e., first pain to heat, mechanical or electrical stimuli. Since A-fibre nociceptors have been shown to be more resistant to a compression/ischemia nerve block than all other A-fibres some of their axons may still conduct after all tested A-fibre related functions (i.e., touch and cold) are blocked . However, in this study A-fibre nociceptors seem not to be the main candidate as the peripheral substrate of SMA because the elevation to vF1/vF10 occurred early during the block when A-fibre nociceptors would be fairly resistant to compression/ischemia. This supports the role of A-beta fibres as peripheral mediators in the von Frey stimulus range used in this study although different receptor organs may be involved, i.e., rapidly (RA) and slowly (SA-I) adapting mechanoreceptors.
Other afferents than low threshold A-beta mechanoreceptive fibres may be implicated regarding DMA in the studied patient group such as nociceptive A-beta fibres [27,28] and A-delta low-threshold mechanoreceptors . The involvement of C-fibre nociceptors with low mechanical threshold  and low-threshold C-mechanoreceptors [31,32] seems less conceivable in this study because C-fibres were unaffected during the continuous nerve block as judged by the preservation of warm perception.
Some methodological considerations deserve attention. The von Frey filaments used in this study did not evoke pain in the contralateral pain free area or in the control area in any patient. However, activation of nociceptive somatosensory channels cannot be ruled out because numerous human and animal studies have shown that the used range of von Frey filaments is sufficient to activate both unmyelinated and myelinated nociceptors, however not necessarily giving rise to pain [29,30,33,34]. In addition, the used range of von Frey filaments increased logarithmically thereby providing a less detailed resolution of measurements in the higher stimulus range, i.e., up to 30 g. Also, the examination of different sensory modalities was made cyclically and approximately every 1–3 min during the nerve block. This range was allowed to secure cessation of stimulus-induced aftersensations in some patients and thus there is a possibility of perception level elevations occurring between examination intervals hence resulting in a recorded value of the time to perception level elevation higher than the true value. Furthermore, an increased perception level early on during the nerve block could be related to disturbances in attention induced by sensations (paresthesias, pain) from the effect of the sphygmomanometer cuff. However, in this study there was no initial increase of temperature perception levels during the first 5 min of the block indicating that distraction from cuff related effects were minor at least in the non-neuropathic skin area.
In conclusion, it is proposed that pain induced by 1 s and 10 s von Frey filament stimulation at perception threshold in patients with neuropathy and DMA/SMA is predominantly mediated by activity in peripheral non-nociceptive A-beta mechanoreceptors. Based on the physiological properties of mechanoreceptors we suggest the involvement of different receptor organs, i.e., rapidly (RA) and slowly (SA-I) adapting mechanoreceptors, in DMA and SMA, respectively. The methods used to assess the perception thresholds of mechanical allodynias deserve further attention as possible adjuncts to suprathreshold stimuli in intervention studies aimed at modifying these stimulus-evoked phenomena.
This study was supported by Grants from Karolinska Institutet.
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About the article
Published Online: 2011-01-01
Published in Print: 2011-01-01
Conflict of interest The authors have no financial or other relationships that might lead to a conflict of interest.