2.2.1 ʻInhibitoryʼ Test Paradigms (ITP).

ITP-studies were characterized by implementation of a noxious or non-noxious inhibitory conditioning stimulus (Fig 2, upper panel; stress-induced analgesia [SIA], spatial summation induced conditioning, diffuse noxious inhibitory control [DNIC], heterotopic noxious conditioning stimulation, conditioned pain modulation [CPM], repetitive noxious stimulation, non-noxious frequency modulated peripheral conditioning and repetitive transcranial magnetic stimulation [rTMS]) [69]. The test-stimulus (Fig 2) was applied heterotopically, at a site different from the site of the conditioning stimulus, or homotopically, at the same site as the conditioning stimulus, where the test stimulus became an integrated part of the conditioning stimulus [19]. The response to the test-stimulus was evaluated by psychophysical measures, e.g., pain ratings, pain threshold and pain tolerance assessments, or physiological measures, e.g., the spinal nociceptive flexion reflex (RIII; Fig 2) [70]. The conditioning inhibitory effect was evaluated by the associated decrease in the response to the test-stimulus: △test-stimulus (Fig 2). MOR-antagonist was administered in order to indirectly uncover an EOS-dependent mechanism in the conditioning response: if the △test-stimulus was attenuated by the MOR-antagonist, a role of the EOS was presumed. In all the studies the outcomes were evaluated against baseline conditions and placebo-controls.

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Fig 2. Schematic illustration of the ʻinhibitoryʼ test paradigms (ITP, upper panel) and the ʻsensitizingʼ test paradigms (STP, lower panel).

The ITP-studies employed an inhibitory conditioning stimulus with evaluation of the associated change in the applied test-stimulus (△test-stimulus). The objective of the ITP-studies was to examine the effect of mu-opioid-receptor (MOR) antagonist on the magnitude of the △test-stimulus, indicating an activation of the endogenous opioid system (EOS) responsible for the conditioning response leading to antinociception/hypoalgesia (the central rectangle [Opioid-dependent mechanism?] indicates a hypothetical augmentation of the conditioning response by the EOS). The STP-studies (lower panel) employed a pain stimulus leading to quantifiable ʻsensitizingʼ CNS-responses, e.g., changes in behavioral measures (hyperalgesia, pain ratings, thresholds, tolerance), nociceptive reflexes, neuroimaging or neuroendocrine variables. In a number of studies a sensitizing conditioning stimulus was applied, e.g., a burn injury [31] and application of capsaicin [35,36], enhancing the nociceptive responses. The objective of the STP-studies was to examine the effect of MOR-antagonist on the magnitude of elicited responses, indirectly either supporting or contradicting an effect mediated by the EOS (the central rectangle [Opioid-dependent mechanism?] indicates a hypothetical attenuation of the response by the EOS). FM Peripheral Conditioning = non-noxious Frequency Modulated Peripheral Conditioning; rTMS = repetitive Transcranial Magnetic Stimulation.

https://doi.org/10.1371/journal.pone.0125887.g002

2.2.2 ʻSensitizingʼ Test Paradigms (STP).

STP-studies were characterized by implementation of a pain stimulus leading to quantifiable, ʻsensitizingʼ, nociceptive responses, i.e., changes in behavioral measures (hyperalgesia, pain ratings, thresholds, pain tolerance), thresholds of nociceptive reflexes, SSEP, or, miscellaneous neuroimaging or neuroendocrine variables (Fig 2, lower panel). In a number of the STP-studies an additional conditioning stimulus was applied, e.g., a burn injury [31] or capsaicin [35,36], enhancing the nociceptive response. MOR-antagonists were administered in order to indirectly uncover an EOS-dependent mechanism in the ʻsensitizingʼ nociceptive response: if the response was enhanced by the MOR-antagonist, an inhibitory role of the EOS was presumed. In all the studies the outcomes were evaluated against baseline conditions and placebo controls.

2.2.3 Habituation and Sensitization.

The phenomenon by which repeated identical stimuli elicit progressively decrements in responses has been operationally defined as habituation [71]. The phenomenon by which repeated identical stimuli elicit progressively increments in responses is here defined as sensitization.

3.2.1 ʻInhibitoryʼ Test Paradigms (25 studies).

The research areas were conditioning modulation models (22 studies) [1–22] and rTMS-models (3 studies) [23–25].

3.2.2 ʻSensitizingʼ Test Paradigms (38 studies).

The research areas were secondary hyperalgesia models (6 studies) [26–31], summation models (2 studies) [32,33], ʻpainʼ models (25 studies) [34–58], nociceptive reflex models (3 studies) [59–61] and miscellaneous (2 studies) [62,63].

3.3.1 ʻInhibitoryʼ Test Paradigms.

Study designs are presented in Table 1. All studies were double-blind and placebo-controlled, and, 17 of the 25 studies [1,2,6,8–10,12,14–17,20–25] were randomized. Four studies reported a counter-balanced design [6,9,20,21], while 19 studies reported use of a cross-over design [1,2,6,7,10–18,20–25]. Three studies, investigating rTMS-induced analgesia, used a sham-control [23–25]. One study used a control 25°C water-immersion test [21].

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Table 1. ʻInhibitoryʼ Test Paradigms: Study Design.

https://doi.org/10.1371/journal.pone.0125887.t001

3.3.2 ʻSensitizingʼ Test Paradigms.

Study designs are presented in Table 2. All of the studies were placebo-controlled while 37 of the 38 studies were double-blind [26–37,39–63]. However, one study [37] mentions only blinding of the subjects, but the study is registered as a controlled clinical trial. Thirty of these studies were randomized [26–32,34–36,39–42,44–52,54–56,58,60,61,63], while 12 studies [35,37,38,41–43,48,50–53,57] used a counter-balanced design. Eight studies did not report a randomized design [33,37,38,43,53,57,59,62].

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Table 2. ʻSensitizingʼ Test Paradigms: Study Design.

https://doi.org/10.1371/journal.pone.0125887.t002

3.4.1 ʻInhibitoryʼ Test Paradigms.

Evaluation was by the Oxford quality scoring system [68] (Table 1). The median (25–75% IQR) score was 2 (2 to 3). Seven out of 25 studies qualified for a score > 2 [8,20–25] and 6 studies for a score < 2 [3,5,6,11,18,19]. In 5 studies either the randomization [22,25] or the blinding procedure [7,13,24,25] was described, but in the remaining 20 studies no information on these procedures were presented. In 5 studies withdrawals and the reasons for withdrawing subjects were reported [8,13,21,24,25].

3.4.2 ʻSensitizingʼ Test Paradigms.

Evaluation was by the Oxford quality scoring system (Table 2) [68]. The median (25–75% IQR) score was 2 (2 to 3). Eighteen of 38 studies qualified for a score > 2 [26,27,30,31,36,39,40,42,44–46,48,50,55,56,58,60,61] and 6 studies for a score < 2 [33,35,37,38,43,59]. In 6 [26,31,39,40,45,58] and 10 [26,31,40,42,46,48,50,55,56,58] studies, respectively, the randomization or the blinding procedure was described. In 26 studies no information on these procedures were presented [27–30,32–38,41,43,44,47,49,51–54,57,59–63]. However, 16 studies reported withdrawals and the reasons for withdrawing subjects [26,27,30,31,36–38,40,44,53,54,57,58,60–62].

3.5.1 ʻInhibitoryʼ Test Paradigms.

None of the 25 studies reported a priori sample size estimations. In 5 studies the confounding issue of limited sample size was discussed [15,16,18,20,22]. Effect size calculations with estimates of Cohen’s d and partial η² (eta squared) [83] were reported in 2 studies [16,22]. In 3 studies corrections for multiple comparisons were made with the Bonferroni adjustment [24,25] and the Tukey-Kramer method [23], respectively. Association was estimated by the Pearson’s correlation coefficient (r) in 10 studies [1,2,4,6,11,14,16,18,21,22]. Analyses of variance (one-way/two-way/three-factor/repeated measures/mixed model ANOVAs) [7,9,12,14,16–19,22–25] or covariance [11] were performed in 13 out of the 25 studies.

3.5.2 ʻSensitizingʼ Test Paradigms.

A priori sample size estimations were reported in 4 [27,31,40,50] of the 38 studies. Post-hoc sample size estimates [40] including analyses with Fisher’s post-hoc least significant difference (LSD) [49,55] were made in 3 studies. In 5 studies the issue of limited sample size was discussed [40,41,50,56,63]. Effect size calculations with estimates of Cohen’s d and partial η² were reported in 2 studies [33,50] and with correlation coefficients in 1 study [41]. In 10 studies corrections for multiple comparisons were made with Bonferroni, Newman-Keul’s multiple range test, Scheffés post-hoc test, Tukey’s test or by applying a 1% significance level [26,28,29,31,32,34,35,48,62,63]. Analyses of variance (one-way/two-way/three-factor/repeated measures ANOVAs; multivariate ANOVA [MANOVA; WILKS test]; linear mixed models; Friedman test) [27–29,32–36,38–40,42,44–53,55–58,60,62,63] were performed in 29 out of 38 studies. Association was estimated by Pearson’s correlation coefficient (r) in 7 studies [28,42,44,45,50–52] and by logistic regression analyses in 1 study [41]. Multiple regression analyses with general linear models (GLM) were made in 2 studies [38,54]. Estimation of significance of indirect effects was made by the Sobel test and by bootstrap estimates [84] in 1 study [42]. Calculations compensating for extreme outliers by Winsorized blockade effect measures were made in 1 study [41].

3.6.1 ʻInhibitoryʼ Test Paradigms.

Demographics are presented in Table 3. The total number of subjects in the ITP-studies was 429, with a median (IQR) number of subjects in each study of 14.0 (8.0 to 24.0). Two studies did not report the gender of the subjects [8,25], but calculated from the remaining 23 studies, the gender ratio (males/females) was 1.9 (249/134). Interestingly, none of the studies rendered information concerning body weight, a detail of some importance, since 11 of the studies used weight-based infusion regimens [5,10,12,16,17,19–21,23–25].

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Table 3. ʻInhibitoryʼ Test Paradigms: Demographics and Drugs.

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3.6.2 ʻSensitizingʼ Test Paradigms.

Demographics are presented in Table 4. The total number of subjects in the STP-studies was 1,048, with a median (IQR) number in each study of 14.5 (11.3 to 23.8) subjects. The second largest (n = 158) [60] and the third largest (n = 151) [61] study reported partially duplicate data [61]. One study [41] was a companion study to a previously published study [85]. Two studies did not report the gender of the subjects [43,48], but based on calculations from the remaining 36 studies, the gender ratio (males/females) was 1.4 (601/430). Only 8 studies rendered information concerning body weight [31,32,34,36,49,51,53] or BMI [56], a detail of some importance, since 9 of the studies used weight-based infusion regimens [28–32,36,49,55,57]. Eight of the studies included patients with fibromyalgia [33,40], chronic low back pain [41,42], borderline arterial hypertension [49], bulimia nervosa [51] or major depression [52], but these data are not presented in the present review.

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Table 4. ʻSensitizingʼ Test Paradigms: Demographics and Drugs.

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3.7.1 ʻInhibitoryʼ Test Paradigms.

Naloxone was used in 24 [1–21,23–25] studies and naltrexone in 1 study [22] (Table 3). Naloxone was administered IV in 21 studies [1–10,12–15,17,19–21,23–25], IM in 2 studies [11,16] and SC in 1 study [18]. In the naloxone studies, estimated from a mean body-weight of the subjects of 70 kg [31] (Table 3), the IV-doses ranged between 6 to 350 microg/kg [14,19] and the IM-doses between 17 to 6,000 microg/kg [11,16]. The estimated weighted mean dose of parenterally administered naloxone was 195 microg/kg. One study used two-doses of naloxone [20]. Naltrexone was administered PO in a dose of 0.71 mg/kg [22]. In all studies normal saline was used as placebo tested against MOR-antagonists.

3.7.2 ʻSensitizingʼ Test Paradigms.

Naloxone was used in 31 studies [26–39,41–49,51,52,54,55,57–59,63] and naltrexone in 7 studies [40,50,53,56,60–62] (Table 4). Naloxone was administered IV in 28 studies [26–34,36–39,41–44,46–49,51,52,54,55,57–59], SC in 1 study [63], PO in 1 study [45] and by iontophoresis in 1 study [35]. In the naloxone studies, estimated from a mean body-weight of the subjects of 70 kg [31] (Table 4), the IV-doses ranged between 6 to 827 microg/kg [27,43,47] and the PO-dose was 457 microg/kg [45]. In one dose-response study target-controlled infusion of naloxone was used [29] in doses ranging from 0.21 to 21 microg/kg. The SC-dose, 1 microg/kg, was minute and only intended for a local effect. The estimated weighted mean dose of IV administered naloxone was 125 microg/kg. Two separate doses of naloxone were used in 6 studies [27,37,39,43,46,47]. One study used a 3-dosing target-controlled infusion regimen [29]. Naltrexone was exclusively administered PO in doses of 0.71 mg/kg [40,50,53,56,60–62]. In all studies normal saline was used as placebo tested across the MOR-antagonists.

3.8.1 ʻInhibitoryʼ Test Paradigms.

Adjuvant drugs were used in 4 studies either due to anxiolytic action (diazepam) [10,12] or to promote induction of pain (capsaicin [Table 3]) [24,25].

3.8.2 ʻSensitizingʼ Test Paradigms.

Adjuvant drugs were used in 11 studies due to the anti-hyperalgesic actions (codeine, fentanyl, ketamine, morphine, paracetamol, oxycodone, remifentanil, tilidine) [26,28,33,42,45,48,50,55,58,62,63] in 4 studies due to the pain-induction ability (capsaicin) [32,34–36] and in 2 studies due to development of opioid-induced hyperalgesia (remifentanil [Table 4]) [28,30].

3.9.1 Electrical Stimuli.

ʻInhibitoryʼ Test Paradigms. Fourteen studies [1–8,10–15] used electrical stimuli as primary test stimuli (Table 5): 9 studies [1,2,5,7,8,10,12,14,15] used transcutaneous stimulation, while 4 studies [3,4,11,13] used non-invasive dental (pulpal) stimulation. Sural nerve-stimulation was used in 6 studies [1,2,10,12,14,15], tibial nerve-stimulation in 2 studies [1,12], alveolar nerve-stimulation in 4 studies [3,4,11,13] and supraorbital nerve-stimulation in 1 study [5]. In 6 studies [1,2,10,12,14,15] the nociceptive flexion reflex (NFR; also termed nociceptive polysynaptic reflex [NPR]) was elicited by sural nerve-stimulation and EMG-recordings of the RIII component from the biceps femoris muscle or the rectus femoris [15]. In 2 of these studies [1,12] the monosynaptic spinal reflex (MSR) was elicited by tibial nerve-stimulation and the EMG-recording of the H-component from the soleus muscle. A detailed description of the characteristics of the electrical stimuli is presented in Table 5.

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Table 5. ʻInhibitoryʼ Test Paradigms: Testing Methods and Results.

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ʻSensitizingʼ Test Paradigms. Eight studies [32,39,43–45,59–61] used electrical stimuli as primary test stimuli (Table 6) and all used transcutaneous stimulation. Sural nerve-stimulation was used in 3 studies [59–61], and additional tibial nerve-stimulation in 1 study [59]. In the former studies the nociceptive flexion reflex was elicited by sural nerve-stimulation and EMG-recordings of the RIII component from the biceps femoris muscle [59–61]. In one of the studies [59] the monosynaptic spinal reflex was elicited by tibial nerve-stimulation and the EMG-recording of the H-component from the soleus muscle. In this study [59] tactile polysynaptic reflexes (PSR) were additionally elicited from sural nerve-stimulation and EMG-recordings of the RII-component from the biceps femoris muscle. A detailed description of the characteristics of the electrical stimuli is presented in Table 6.

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Table 6. ʻSensitizingʼ Test Paradigms: Testing Methods and Results.

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3.9.2 Mechanical Stimuli.

ʻInhibitoryʼ Test Paradigms. One study used pin-prick stimulations by nylon filaments [19] with bending forces from 0.08 mN to 2,492 mN [19] for assessments of detection thresholds (Table 5). One study used isometric handgrip strength measured by dynamometry [9].

ʻSensitizingʼ Test Paradigms. Innocuous stimuli with brush [26], cotton-wool [28,29], or gauze swabs [27] were used for assessment of allodynia (Table 6). Pin-prick stimulations used in 8 studies, were by nylon filaments [26–29,31,34,63] with bending forces 121 mN to 1,237 mN [34,63], or a non-flexible steel wire [30], and, were used for assessments of secondary hyperalgesia areas [26–31], pain thresholds [31,34] and pain ratings [63]. Pressure algometry was used in 4 studies for assessments of pain thresholds [34,40] and suprathreshold pain ratings [27,41]. Handgrip strength was measured by dynamometry in 8 studies [37,38,41,42,46–48,50] and skin pinching in 1 study [49]. Vibratory stimulation was used in 1 study [27].

3.9.3 Thermal Stimuli.

ʻInhibitoryʼ Test Paradigms. Contact thermodes were used in 9 studies [4,16,19–25] with contact areas ranging from 1.0 cm² to 9.0 cm² [20,23] (Table 5). In 2 studies [9,18] radiant heat by a halogen globe directed at the skin, was used. Thermal thresholds were assessed in 7 studies [4,16,18–20,23,24] and thermal pain ratings in ten studies [9,16,17,19–25]. Eight studies used either phasic [16,19–21,23,24] or tonic heat stimuli [9,22,24,25], with temperatures ranging from 46°C to 50°C [22], stimulus duration of 5 [21] to 30 s [22] and repeated heat stimuli from 5 [22] to 64 times [21]. In 2 studies [16,19] temporal summation of heat stimuli in trains was used. In one of these [19] identical heat stimuli were used both as test stimulus and conditioning stimulus, in single mode and repeated mode, respectively. In 4 studies [9,15–17] cold-water immersion tests were used. In 1 study an ascending and descending spatial summation paradigm [17] was used, and in 2 studies [16,17] the cold-water immersion tests was employed both as test stimulus and conditioning stimulus. Two studies [24,25] used a block-testing technique, assessing heat allodynia with 3 separate 22 s stimuli using a temperature of the contact thermode calibrated at baseline corresponding to a pain rating of 7 (NRS, 0–10).

ʻSensitizingʼ Test Paradigms. Contact thermodes were used in 15 studies [26,27,31,33,36,40,42,51–55,57,58,62] with the contact areas of the thermodes ranging from 2.0 cm² to 12.5 cm² [26,42] (Table 6). Three studies [35,39,63] used a source of radiant heat, in 2 of the studies by a halogen globe with apertures ranging from 94 mm² to 1 cm² [35,63]. Thermal thresholds were assessed in 12 studies [26,27,31,35,39,40,42,51–53,55,63] and thermal pain ratings in 7 studies [27,33,36,42,57,62,63]. Ten studies used phasic [33,39,42,51,53,57,62,63] and/or tonic [36,51,54] heat stimuli, with temperatures ranging from 43°C [57] to 51.8°C [58], stimulus duration of 0.7 s [33] to 35 s [51] and repeated up to 10 times [57]. In 1 study [33] identical heat and cold stimuli were used both as test stimulus and conditioning stimulus, in single mode and repeated mode, respectively. Cold-water immersion tests (0°C to 10°C) with pain ratings were used in 4 studies [37,38,53,56].

3.10.1 Electrical Stimuli.

ʻInhibitoryʼ Test Paradigms. Six studies [1,2,7,10–12] used noxious electrical conditioning stimuli and 5 studies [3,5,6,13,20] used non-noxious electrical stimuli (peripheral conditioning). The studies used transcutaneous sural nerve-stimulation [1,2,10,12,20], transcutaneous nerve-stimulation [3,6,7,13], high-frequency (100 Hz) stimulation [3,5,20], or, low-frequency stimulation [13], or repetitive (1 Hz) dental stimulation [11]. Detailed stimulation characteristics are presented in Table 5.

ʻSensitizingʼ Test Paradigms. Three studies used noxious electrical conditioning stimuli applied intradermally [28–30] or transdermally [28] (Table 6). Data from the 3 studies [59–61] using a classical electrical DNIC-paradigm are reported in paragraph 3.9.1, second subsection and Table 6.

3.10.2 Mechanical Stimuli.

ʻInhibitoryʼ Test Paradigms. Two studies [4,9] used the modified, ischemic submaximal effort tourniquet test, with assessment of hand grip strength, as conditioning stimulation (Table 5). One of these studies in addition used exercise (6.3 mile [10 km] run) at 85% of maximal aerobic capacity as a physiological conditioning stressor [9]. Another study employed 20 min leg and arm conditioning exercises on ergometers [13].

ʻSensitizingʼ Test Paradigms. Six studies [37,41,42,46–48] used the modified, ischemic submaximal effort tourniquet test, with assessment of hand grip strength, as conditioning stimulation (Table 6).

3.10.3 Thermal Stimuli.

ʻInhibitoryʼ Test Paradigms. Nine studies [8,14–19,21,22] used thermal stimuli as a conditioning stimulus (Table 5). Cold-water-immersion tests (0 to 12.9°C) [8,15–18,21,22] and hot-water-immersion test (46°C) [14] were used in 8 studies. Two studies used cold-water-immersion tests (1 to 3°C) repeated 4 to 10 times [16,18]. Repeated heating, using a temporal summation pattern was used in 1 study [19].

ʻSensitizingʼ Test Paradigms. Six studies [26,27,31,33,62,63] used thermal stimuli as a conditioning stimulus (Table 6). First degree burn injuries, leading to development of erythema and hyperalgesia, were induced by thermodes [26,27,31] (area 12.5 cm², 47°C, 7 min), heat probes [63] (area 0.8 cm², 48°C, 2 min) and UV-light [62] (150W xenon lamp, UV_A [400 nm] and UV_B [290 nm], aperture 2 cm), in 5 studies. Repeated suprathreshold heat stimuli and infrathreshold cold stimuli, respectively, attaining a temporal summation pattern, were employed in 1 study [33]. A brief conditioning sensitization heat stimulus (brief thermal sensitization) was used in 1 study [31].

3.10.4 Pharmacological Stimuli.

ʻInhibitoryʼ Test Paradigms. Conditioning with capsaicin 0.1% cream applied 30 min prior to heating with a contact thermode was used in 2 studies [24,25] (Table 5).

ʻSensitizingʼ Test Paradigms. Conditioning procedures with capsaicin administered SC (10 microg) [32], IM (50 microg) [34], and, topically, in an aqueous solution (0.4 ml, 0.6%) [35] or cream (35 mg, 10%) [36], were used in 4 studies (Table 6). Two of these studies used heat to further sensitize the capsaicin treated areas [35,36].

3.10.5 Miscellaneous Stimuli.

ʻInhibitoryʼ Test Paradigms. In 3 studies [23–25] rTMS was applied as a conditioning analgesic stimulus. In the 2 latter studies [24,25] rTMS was targeted at the left dorsolateral prefrontal cortex (DLPFC) while in the third study [23] the analgesic effects were targeted at the right motor cortex (M1) and right DLPFC (Table 5).

3.11.1 ʻInhibitoryʼ Test Paradigms.

Conditioning Modulation Models. The primary objectives of the conditioning modulation studies are schematically presented in Fig 2A (upper part) and summarized in Table 1. All studies used a paradigm where the reversal effect of MOR-antagonist across 5 apparently different conditioning models (as defined by the authors) was examined. The conditioning models included stress-induced analgesia (7 studies) [1,2,7–10,12], spatial summation induced conditioning (1 study) [17], diffuse noxious inhibitory control (DNIC; 4 studies) [4,14–16], heterotopic noxious conditioning stimulation (1 study) [21], conditioned pain modulation (CPM [heterotopic noxious conditioning stimulation and DNIC are by some authors^16;38 included in the CPM-moiety; 1 study) [22], repetitive noxious stimuli (3 studies) [11,18,19] and non-noxious frequency modulated peripheral conditioning (5 studies) [3,5,6,13,20].

The outcome variables affected by the conditioning stimulation were pain perception [4,7,9,10,12,15–23,25], pain thresholds [3,4,6,8,11,13,16,18,19], nociceptive reflex thresholds (RIII) [1,2,10,12,14,15], autonomic parameters (heart rate, respiratory rate, arterial blood pressure) [1,2,12,16], sensory detection threshold [18], MRT (blood oxygen level dependent [BOLD])-responses [21], nociceptive component of blink-reflex (R2) [5], psychometrics [9,22] and SSEP [7]. The test stimuli were applied heterotopically [4–6,8,14–16,21], homotopically [1,2,7,10,11,17,18,20] or, both heterotopically and homotopically [3,9,12,13,19,22]. The outcomes of MOR-antagonist administration were reversal of the effects of the conditioning modulation (Fig 2), examining relative increases in pain perception, pain threshold, nociceptive reflex threshold or autonomic variables.

Repetitive Transcranial Magnetic Stimulation Models. The primary objective of the dorsolateral prefrontal cortex (DLPFC) targeted rTMS-studies was to examine the effect of a MOR-antagonist on stimulation-evoked analgesia [23–25] (Table 1). The test stimuli were applied homotopically, i.e., at the right side when rTMS was targeted at the left hemisphere and vice versa. The outcomes were changes in pain perception, pain threshold and pain tolerance (Table 5).

3.11.2 ʻSensitizingʼ Test Paradigms.

Secondary Hyperalgesia Models. The objectives were to examine if administration of a MOR-antagonist was associated with an increase in secondary hyperalgesia areas induced by a thermal injury [26,27,31], thermal suprathreshold stimulation [31] or noxious electrical high-intensity stimulation [28–30] (Table 2). Three studies examined either the effect of naloxone on ketamine-induced secondary hyperalgesia area [26] or opioid-induced hyperalgesia following remifentanil [28,30]. In both of these studies only data pertaining naloxone vs. placebo administration were considered relevant for this review. Another study examined re-instatement of secondary hyperalgesia area 72 hours after the burn-injury [31]. Outcomes were changes in secondary hyperalgesia areas, i.e. allodynia- or hyperalgesia-areas, evaluated by tactile or pin-prick stimuli, respectively (Table 6).

Summation Models. The objectives of the 2 summation studies were to examine the effect of a MOR-antagonist in regard to spatially directed expectation of pain [32], or temporal summation of phasic heat- and cold-stimuli [33] (Table 2). In the former, largest study (n = 173) of the review [32], SC injections of capsaicin were administered, unilaterally in the hand or in the foot, and expectations of analgesia were induced by application of a placebo cream, told to contain a ʻpowerful local anesthetic substanceʼ. This paradigm induced a placebo response in the treated body part, but not in untreated body parts. The objective of the study was to examine reversal effects of a MOR-antagonist on the placebo-response. In the latter study [33], repeated heat and cold stimuli, induced central sensitization, and the objective was to study the differential effect of naloxone on the first (single thermal stimuli [A-delta]) and second pain (repetitive thermal stimuli [C-fiber]) in the temporal summation process. The outcome parameters were in both studies pain ratings (Table 6).

ʻPainʼ Models. The objectives of these 25 studies were to examine the effect of MOR-antagonists on pain induced by capsaicin [34], capsaicin kindled by heat [35,36], cold [40,52,55], cold pressor test [37,38,53,56], electricity [39,43–45], heat [39,40,42,51–55,57,58], ischemia [37,42,46–48], or mechanical stimuli [37,38,41,42,46,47,49,50] (Table 2). The outcomes of MOR-antagonist administration compared to placebo, were changes in sensory thresholds [52], pain ratings [34,36–38,41,42,44–50,53–57], pain thresholds [34,35,39,40,42,43,51–53,55], pain tolerance [40–43,53], psychometrics [37,41,46,47], neuroimaging techniques [54,57], SSEP [44,45,58] or autonomic variables [38,49,50] (Table 6).

Nociceptive Reflex Models. The common objective of these 3 studies [59–61] was to examine the effect of MOR-antagonists on the thresholds of nociceptive flexion reflexes (Table 2). In addition, the first study [59] investigated non-nociceptive spinal reflexes, while the 2 other studies [60,61] investigated pain thresholds and pain ratings. The outcome parameters were nociceptive sural nerve stimulation induced changes in the biceps femoris muscle EMG-RIII component (Table 6).

Miscellaneous. The objective in the 2 studies was to examine the effects of MOR-antagonist on opioid induced antihyperalgesia following a burn-injury [62,63]. In both studies the MOR-antagonist was administered prior to opioid. The outcomes were pain ratings [62,63] and pain thresholds [63].

3.12 Secondary Objectives. Secondary objectives related to the administration of MOR-antagonists are presented in Tables 1 and 2.

3.13.1 ʻInhibitoryʼ Test Paradigms.

Conditioned Modulation Models. In the 7 SIA-studies [1,2,7–10,12], naloxone reversed the antinociceptive or analgesic effects of the conditioning stimuli, substantiated by decreases in threshold of the nociceptive flexion reflex (RIII) [1,2,10,12], threshold of the monosynaptic spinal reflex [1,12], electrical pain thresholds [8], and, increases in SSEP [7] and pain ratings (Table 5) [7,10,12]. One study demonstrated complete reversal of post-exercise ischemic hypoalgesia [9], although no response of naloxone to post-exercise thermal hypoalgesia was seen.

In the spatial summation induced conditioning study [17] naloxone reversed the spatial summation induced activation of the endogenous pain inhibitory system.

In 2 [14,16] of the 4 DNIC-studies [4,14–16], a naloxone-dependent complete reversal of the DNIC-induced increase in nociceptive flexion reflex [14] and an increased cardiovascular reactivity to tonic noxious cold stimulus [16] were demonstrated. However, in the latter study [16] no effect of naloxone on the heat pain perception (DNIC-efficiency [86]), compared to placebo, was observed. In the two remaining studies the findings were ambiguous [4,15]. In one of these studies [4] a significant naloxone-dependent effects were not demonstrated, though a likely reversal effect of naloxone on the conditioning-induced increase in heat pain thresholds was seen. In the other study [15] a trend (P = 0.07) towards a naloxone-dependent blocking effect of DNIC was observed and the authors attributed this to a type II error. The sample size in this cross-over study was 20 subjects (subgroup of extensive metabolizers of sparteine) indicating that the naloxone-dependent effect in this study, probably was rather weak.

In the heterotopic noxious conditioning stimulation study [21], naloxone generally did not affect pain ratings to phasic heat test stimuli during the cold-water immersion test (CWIT), however increased pain ratings to the tonic CWIT-conditioning stimulus were observed, compared with placebo treatment. The study also demonstrated an impaired correlation between the CWIT-pain ratings and the measure of endogenous analgesia (assessed by the phasic heat stimuli) in the naloxone-sessions compared to the placebo-sessions.

In the single CPM-study [22] (Table 5), it was demonstrated that naltrexone abolished a CPM-induced decrease in heat pain ratings, but only in subjects with low ratings on the pain catastrophizing scale (PCS).

The 3 studies [11,18,19] employing repetitive noxious stimulation, utilized very different methodological designs and reached different conclusions. One study observed that local administration of naloxone was associated with augmented sensitivity during repeated CWIT [18], while the other studies [11,19] were unable to demonstrate any naloxone-dependent effects on electrical pain thresholds [11] or on the magnitude of habituation in a complex model of repeated heat stimuli [19].

In 3 out of 5 studies [3,5,6,13,20] utilizing a non-noxious frequency modulated peripheral condition stimulation model, no effect of naloxone on the nociceptive component of the blink reflex [5] or dental electrical pain thresholds [3,6] was seen. In one study a paradoxically prolonged increase in the electrical dental pain threshold was observed after naloxone administration [13], indicating a hypoalgesic effect. A study using high-frequency transcutaneous electrical nerve stimulation induced thermal hypoalgesia was not affected by placebo or low-dose naloxone (0.04 mg/kg), but was blocked with high-dose naloxone (0.28 mg/kg) [20].

In summary, 8 studies [1,2,7–10,12,17], demonstrated consistently for SIA and for spatial summation induced conditioning, that conditioning effects were reversed by naloxone, corroborating a role of the endogenous opioid system in these testing models. For the remaining models [3–6,11,13–16,18–22] ambiguous findings on the MOR-antagonist efficacy in reversing the conditioning effects were reported.

Repetitive Transcranial Magnetic Stimulation Models. In these interesting sham-controlled studies with rTMS targeted at the dorsolateral prefrontal cortex (DLPFC) [23–25] and the motor cortex (M1) [23], pre-treatment with naloxone attenuated the analgesic effect indicating an endogenous opioid-dependent mechanism of rTMS (Table 6). Two of the studies [24,25] observed significant attenuating effects of naloxone on DLPFC-targeted rTMS, while the third study [23] only observed this for M1-targeted rTMS, but not for DLPFC-targeted rTMS.

3.13.2 ʻSensitizingʼ Test Paradigms.

Secondary Hyperalgesia Models. One study [29] demonstrated a dose-dependent naloxone response, with increasing magnitudes of secondary hyperalgesia areas induced by high intensity intradermal electrical stimulation. Interestingly, in 2 [28,29] of 3 studies [28–30] with intradermal electrical stimulation increased pain ratings, indicating heightened pain sensitivity, were observed following naloxone administration. However, in 5 [26–28,30,31] of the 6 secondary hyperalgesia area studies [26–31] no signs of naloxone-dependent increases in secondary hyperalgesia areas were observed (Table 6). However, one of the studies examined the late reinstatement of secondary hyperalgesia areas, 72 hrs after a mild burn-injury [31].

Summation Models. In the large placebo study [32] naloxone completely abolished the placebo response indicating that endogenous opioids, spatially modulate specific placebo responses. In the temporal summation study [33] using repeated phasic heat and cold stimuli, no effect of naloxone, compared to placebo, on thermal wind-up, “first” pain or “second” pain, was observed (Table 6).

ʻPainʼ Models. In the 18 studies [34,36–38,41,42,44–50,53–57] using pain ratings as primary outcomes, 6 studies [36,44,49,53,54,57] observed an MOR-antagonist dependent effect, while 10 studies did not observe any significant effect [34,37,38,45–48,50,55,56] (Table 6). Five studies, examining the response to MOR-antagonists, reported increases in ratings to electrical stimuli (only in pain-insensitive individuals) [44], non-noxious heat [57], noxious heat [36,54], and skin pinch [49], while 1 study reported paradoxically decreased pain ratings during the cold pressor test [53]. Thirteen studies did not report any MOR-antagonist dependent changes in pain ratings in regard to the cold pressor test [37,38,56], dynamometric tests [37,38,46,47,50], electrical stimuli [45], ischemia [41,42,48], phasic thermal stimuli [42,53,55], or, pressure and pinprick stimuli [34].

In regard to thermal thresholds 2 studies, assessing heat pain thresholds,[35,38] observed an increase in thresholds, following either local, iontophoretic [35] or systemic [38] administration of MOR-antagonist, indicating a hypoalgesic mechanism of action. However, 6 studies [40,42,51–53,55] could not substantiate any change in heat pain thresholds associated with administration of MOR-antagonist. Three of these studies [40,52,55], additionally, could not show any effects on cool detection, cold pain or cold tolerance thresholds. Two studies [39,43] examining electrical thresholds and 1 study examining vibratory thresholds [52] could not show any effect related to the systemic opioid-blockade. Three studies measured SSEP [44,45,58], but only in 1 study [45] MOR-antagonist dependent changes in SSEP were observed. In one [49] of 2 studies [49,50] using microneurographic recordings of sympathetic nerve activity (MSNA), an increase in activity related to the opioid-blockade was seen.

Interestingly, 2 neuroimaging studies [54,57], using fMRI [54] and the BOLD-contrast imaging technique [54,57] could substantiate functional changes, i.e., increases in activity of several brain regions [54] or blocked deactivation of the pregenual ACC (anterior cortex cinguli) [57], correlating with perceptual responses to noxious and non-noxious heat exposure, following administration of MOR-antagonist (Table 6).

Summarizing the results from the 25 studies, the direction of MOR-antagonist dependent effect on pain ratings, threshold assessments and SSEP appear quite ambiguous and inconsistent [42]. Any evidence for stimulation modality specific changes in response to MOR-antagonists is lacking. However, the results on heat stimuli from the 2 neuroimaging studies seem consistent and promising [54,57].

Nociceptive Reflex Models. In the first study [59] naloxone facilitated, i.e. increased the amplitude, of the monosynaptic spinal reflex, but did not affect the tactile polysynaptic reflex (RII). In 2 [59,60] of the 3 threshold studies [59–61] no effect of naloxone on the threshold of nociceptive flexion reflex (Table 6) could be demonstrated. In the third study [61] the results were somewhat at odds with previous findings, indicating lowered nociceptive flexion reflex activity (defined as the rectified biceps femoris EMG measured at the 90–150 ms post-stimulation interval) after administration of naltrexone: a hypoalgesic effect corroborated by the naltrexone-associated findings of significantly decreased pain ratings at electrical pain and tolerance thresholds. However, both in the second [60] and third study [61] administration of naltrexone during noxious sural nerve-stimulation was associated with increased pain in female subjects, while in male subjects naloxone administration was associated with an increase in electrical pain thresholds.

Miscellaneous Models. In the 2 burn-injury studies [62,63] a naloxone-dependent reversal of opioid-induced anti-hyperalgesia was demonstrated primarily by an increase in heat sensitivity (Table 6).

3.14.1 ʻInhibitoryʼ Test Paradigms.

Seven of the 24 studies described either drug-related adverse effects [1,22], withdrawals not related to administration of MOR-antagonist [13,21,24,25] or the occurrence of outliers [8]. In one study [22] adverse effects, termed “mild side effects”, like mental dulling, confusion, sedation and poor balance were reported. Unfortunately this study only described mean values of the adverse effects based on the group, while the absolute number of subjects experiencing the adverse effects was not given. In the study with the highest naloxone dosis, i.e. 6,000 microg/kg, the subjects were unable to tell the correct order (with a likelihood higher than chance) of active drug vs. placebo and no adverse effects were reported [16].

3.14.2 ʻSensitizingʼ Test Paradigms.

Thirteen of the 38 studies described either occurrence of drug-related adverse effects [26,27,38,39,42,51,62], withdrawals not related to administration of MOR-antagonist [30,36,38,53,57,58] or the occurrence of outliers [55]. Six subjects were reported to experience adverse effects: 1 subject had psychotropic effects due to ketamine [26], 1 subject developed a second degree burn injury [26] and 4 subjects experienced sensation of warmth, palpitations, drowsiness, nausea and vomiting [27,62]: in 3 of the subjects very likely related to administration of MOR-antagonist. In one study [39] the responses in the side effect questionnaires showed that tiredness, lightheadedness, nausea, abdominal “grumbling”, and mood changes were reported slightly more often after naloxone than after placebo. In another study [51] a "drowsiness" scale demonstrated higher values for naloxone than for placebo.

4.2.1 ʻInhibitoryʼ Test Paradigm.

The descending conditioned pain modulation system (DNIC or CPM; cf. 4.4.2) is considered an important factor regulating pain sensitivity in humans [87–89] and it has been suggested that pathological changes in the CPM-system are important for the development of chronic pain in chronic tension headache, fibromyalgia and persistent postoperative pain [33,88,90–94]. The CPM system is in part modulated by exogenous opioids: a sub-therapeutic dose of morphine may uncouple the conditioning system, deregulating the balance between pain sensitization and inhibition [14,95]. Impairment of the descending inhibitory systems, e.g., the EOS and CPM, may contribute to the trajectory from acute to chronic pain. Research in blockade of the opioid system may improve our understanding of the underlying pathophysiological mechanisms and may lead to a reformulation of strategies for the prevention and management of chronic pain.

4.2.2 ʻSensitizingʼ Test Paradigm.

Obviously, a number of scientific issues are common for the complementary test paradigms, ITP and STP, however, injury or disease related nociceptive input to the CNS may trigger a sustained excitability and increased synaptic efficiency in central neurons [93], i.e., central sensitization (CS), a stimulus-response enhancing mode which may contribute to the development and maintenance of a chronic pain state [93,96]. Animal data suggest that CS outlasts overt signs of hyperalgesia, in a silent form termed ʻlatent sensitizationʼ (LS). The LS far outlasts the conventional duration of the injury assessed by behavioral measures, but can be unmasked by administration of a centrally acting MOR-antagonist leading to “rekindling” or reinstatement of hyperalgesia [92,93,97]. Thus, post-injury pain remission is maintained in part by the EOS that masks the pro-nociceptive components of LS. ʻLatent sensitizationʼ could prime central nociceptive circuitry such that, when inhibitory systems fail, as upon exposure to excessive stress, a pain episode ensues [93,97]. The latent predisposition to relapse, may explain the episodic nature and vulnerability to stressors that accompany chronic pain states in humans [93,97].

4.4.1 Stress-induced Analgesia.

In the SIA-models two stimuli are required: an aversive stressor-stimulus and a noxious test-stimulus [99]. The aversive stimuli in the present SIA-studies [1,2,7,8,10,12], were intense, noxious electrical conditioning stimulation [1,2,7,10,12], (in 4 of the studies even termed ʻinescapableʼ shocks [1,2,10,12]), the cold pressor test [8], an arithmetic stressing test or a 6.3 mile run at 85% of maximal aerobic capacity [9] (Table 5). In the electrical studies an alternating procedure was used, randomizing sequences of high-intensity noxious and low-intensity tactile stimuli, in order to facilitate apprehension and discomfort, adding psychological factors to the test paradigm. Electrical noxious test-stimuli evaluating the conditioning effects were employed in all studies.

Transcutaneous electrical stimulation is a ubiquitous method used in pain research, due to its ease of use, flexibility, and in particular, due to its versatility, regarding stimulation rates, intensity adjustments and generation of sequence-patterns. The method elicits pain and hyperalgesia by direct axonal stimulation, bypassing the sensory nerve endings [100]. Electrical stimuli are thus probably more suitable for examination of central pain components, than ʻphysiologicalʼ thermal and mechanical stimuli, reflecting both peripheral and central components of the pain response [101]. High-intensity, noxious electrical stimulation (for stimulation characteristics cf. Table 5) is associated with activation of the EOS and an apparent stimulation-dependent decrease in pain ratings [28,29,102]. The degree of habituation, assessed as decrements in pain ratings, following 45–180 min continuous noxious electrical stimulation, is between 20–60%, using constant stimulation intensity [28,29,100,102,103]. Using a test paradigm, adjusting the current strength to a constant level of pain perception, the increase in stimulation intensity during 45 min of continuous stimulation, is 260% [103]! Thus, in the SIA-studies the decrements in pain ratings [7,10,12], increases in threshold of the nociceptive flexion reflex (RIII) [1,2,10,12], thresholds of the monosynaptic spinal reflex [1,12], electrical pain thresholds [8], and, decreases in SSEP [7], observed in controls, could in part be explained by habituation [7] (Table 5).

Prolonged and intense, noxious electrical conditioning stimulation activates a naloxone-sensitive (indicating recruitment of the EOS) and a naloxone-insensitive inhibitory system [29]. Interestingly, these findings are not modality specific, but also apply to noxious contact heat [104,105], laser stimuli [106] and capsaicin application [36].

In heat-models it has been shown, that habituation involves the descending antinociceptive system and in addition comprises a naloxone-insensitive component [19]. The fMRI-based studies demonstrated that part of the antinociceptive system, the rostral and subgenual anterior cingulate cortex (rACC/sgACC)) and the periaqueductal grey area, were involved in habituation, indicating a contribution of central pathways to the phenomenon of habituation [104,106].

Experimental factors that potentially may influence habituation and its central correlates are the stimulation modality, stimulation rate, the timeline of the stimulation, i.e. short-term or long-term habituation, and, the use of phasic or tonic stimulation patterns [19,103,104].

4.4.2 Diffuse Noxious Inhibitory Control and Conditioned Pain Modulation.

DNIC and CPM are synonymous terms, and it has recently been recommended that DNIC should be reserved for animal and CPM for human research [88]. In the present review the terms were used explicitly as stated in the studies.

The 4 DNIC-studies [4,14–16] presented similar findings in regard to the ʻclassicalʼ DNIC-paradigm: a high intensity noxious stimulus decreased the response or increased the threshold to a heterotopically applied pain-stimulus [87]. However, naloxone-induced reversal of the DNIC-effect was only unambiguously demonstrated in one of the studies [14], an effect that would have been anticipated since previous studies have indicated opioid-sensitive components of DNIC [14,87,107]. Among the 3 DNIC-studies [4,15,16] with ambiguous findings, two of the studies were low-powered [4,16] and the authors of one of the studies stated that the negative findings could likely be attributed to a lack of statistical power (n = 6) [16]. Interestingly, one of the studies [15], seemingly adequately powered, indicated a weak naloxone-dependent effect (cf. 3.13.1).

Although the CPM-study [22] partially demonstrated a naltrexone-dependent abolishment of the conditioning induced decrease in pain response (Table 5), the general ambiguous findings in the 5 studies [4,14–16,22] seem difficult to reconcile.

4.4.3 Non-noxious Frequency Modulated Peripheral Condition Stimulation.

The 5 studies on non-noxious frequency modulated peripheral conditioning did not show any consistent sign of involvement of the EOS [3,5,6,13,20].

4.4.4 Repetitive Transcranial Magnetic Stimulation.

The methodological qualities of these studies were among the highest in the present review. An advantage was that the studies used sham-controlled and placebo-controlled procedures. Two of the studies [24,25] observed significant attenuating effects of naloxone on DLPFC-targeted rTMS, while the third study [23] only observed this for M1-targeted rTMS. The studies utilized thermal test paradigms, although they differed in regard to pretreatment with capsaicin that was used in two of the studies [24,25], i.e., application of capsaicin may lead to more intense pain perception [108]. The studies also differed in regard to left [24,25] and right [23] hemisphere targeted stimulation.

4.5.1 Secondary Hyperalgesia Models.

One study [29] demonstrated a dose-dependent naloxone response, with increasing magnitudes of secondary hyperalgesia areas induced by noxious, high intensity, intradermal electrical stimulation. Interestingly, in a preceding study by the same research group [28], using a nearly identical set-up, naloxone-infusion 10 microg/kg, was associated with a highly significant increase in secondary hyperalgesia area (140%) compared to pre-infusion values (P < 0.01). However, since the baseline values successively increased during the 30 min infusion period, the increase in secondary hyperalgesia areas compared to controls, did not reach significance (P = 0.16; deviations in baseline assessments in noxious electrical stimulation, cf. 4.4.1). The study abstract correctly indicated that naloxone resulted in increased pain ratings (ʻantianalgesiaʼ; P < 0.001), but, erroneously indicated that naloxone resulted in ʻmechanical hyperalgesia (P < 0.01)ʼ [28]. The third secondary hyperalgesia study using high intensity intradermal electrical stimulation, on remifentanil-induced opioid hyperalgesia (OIH) [30] was not able to demonstrate any effect of naloxone on secondary hyperalgesia. Though the study generally had a double-blind, controlled and randomized design, the naloxone part of the investigation did not include a placebo arm per se, but naloxone was administered blinded to the remifentanil and the placebo groups.

Consequently, in 5 of 6 secondary hyperalgesia studies obvious signs of EOS involvement could not be demonstrated, however, naloxone-dependent hyperalgesic responses during high intensity noxious electricity stimulation cannot be excluded. Recent data, examining latent sensitization [109] using a burn injury model [31] indicated that administration of a high dose of naloxone 2 mg/kg, 1 week after the injury, in 4 out of 12 subjects seem to be associated with late re-instatement of secondary hyperalgesia areas (submitted: Pereira MP et al. ʻEndogenous opioid-masked latent pain sensitization: studies from mouse to human.ʼ).

4.6.1 General Issues.

The bibliographic age and the exploratory nature of the studies should be taken into consideration when discussing study bias, i.e., the risk that the true intervention effect will be overestimated or underestimated (http://handbook.cochrane.org/ [accessed 07.24.2014]). The present review using the simple Oxford quality scoring system from 1996 [68] demonstrated a high likelihood of bias, due to inaccurate reporting of randomization and blinding procedures. The Oxford quality scoring system was chosen in respect of the bibliographic age of the studies since it presents a more lenient evaluation paradigm than more sophisticated methods like the Cochrane Collaboration’s tool for assessing risk of bias. Approximately 25% and 50% of the studies were published before 1984 and before 2000, respectively, while the early report on the Consolidated Standards of Reporting Trials (CONSORT) was published in 1996 [110], with revised versions 2001 [111] and 2010 [112]. Compliance to these standards has been a requirement for randomized controlled trials (RCTs) in a number of clinical journals for more than 17 years [110]. The CONSORT statement thus contains guidelines applicable to clinical RCTs, but is it relevant for experimental RCTs?

The editorial accompanying the first CONSORT publication stated: “It seems reasonable to hope that, in addition to improved reporting, the wide adoption of this new publication standard will improve the conduct of future research by increasing awareness of the requirements for a good trial. Such success might lead to similar initiatives for other types of research”[110]. Requirements for clinical RCTs as outlined in CONSORT and SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) [113], could also be considered essential for experimental research in order to heighten validity, reliability and reproducibility of data, facilitating accurate reporting, evaluation and interpretation of study-data. Guidelines for reporting animal research data, ARRIVE (Animals in Research: Reporting In Vivo Experiments) [114], based on the CONSORT criteria, have been published and even extrapolated for use in a review including human experimental research [115].

4.6.2 Statistical Issues.

The heterogeneity of the statistical methods used was considerable. Various ANOVA-methods, some rather advanced [24,42], were used in 41/63 studies, while a priori and post-hoc sample size estimations were only employed in 4/63 and 3/63 studies, respectively. Furthermore, in 49/63 studies, statistical methods aimed at reducing the risk of type I error (α [false positive]) associated with multiple comparisons, were not applied. It can readily be calculated that the likelihood by chance of achieving one or more false positive results, presenting as significant values (P < 0.05), during 5 and 10 uncorrected pair-wise comparisons, which was a normal procedure in the studies, are 23% and 40%, respectively, giving a high likelihood for falsely rejecting the null hypothesis [116,117]. A simple measure to attenuate the risk is to decrease the significance level to 1% which would give corresponding likelihoods of less than 4.9% and 9.6%, respectively. Conversely, the risk of committing a type II error (β [false negative]), i.e., indicating the power of the study, was generally not reported in the studies, although the limited sample size was discussed in 10/63 studies.

However, it could be argued that all the reviewed studies are experimental, and as such exploratory and hypothesis-generating in nature. In studies of healthy individuals, inferences from qualitative aspects, within-subject variances and fixed-effect models are often more important than inferences from quantitative aspects, between-subject variances and random-effect models, the latter being preferred in clinical research examining groups of patients [118]. The number of subjects needed is obviously much smaller for experimental research compared to clinical studies, mainly due to a much larger inherent biological variance in patients compared to healthy subjects. However, the exploratory nature of an experimental study is sometimes used as a an excuse for not adhering strictly to common statistical requirements [117]. For all research, decisions on the null hypothesis, primary and secondary outcomes, and, estimations of outcome variability (from pilot-data if not available from literature), minimal relevant differences, sample size and effect size calculations should be stated even in studies of exploratory nature. Otherwise, there is an obvious risk of wasting valuable research time and efforts, leading to ethical, economical or scientific dilemmas, which might impede future research [116,117,119].

4.6.3 Methodological Issues.

Lack of standardization across the studies and the stimulation methods are evident (Tables 5 and 6). Guidelines for sensory testing procedures have been presented [120–123], but standardized protocols, like the German Research Network on Neuropathic Pain (DFNS) [122–124] were not used in any of the studies. Aspects of data reproducibility and validity were only discussed in few studies [29,31,125].