YNIMG-11841; No. of pages: 6; 4C: 2, 3, 4 NeuroImage xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

NeuroImage

BOLD responses to itch in the human spinal cord

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Missanga F. van de Sand ⁎, Christian Sprenger, Christian Büchel

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Department of Systems Neuroscience, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany

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Article history: Accepted 5 December 2014 Available online xxxx

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Keywords: Itch Spinal cord Histamine Spinal fMRI Spinal cord processing

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Itch is an independent sensory modality and a very common symptom with manifold causes. However, the neuronal representation of itch perception in the central nervous system is not entirely understood and there is hardly any knowledge about neuronal correlates of itch in the human spinal cord. In the present study we aimed to identify itch-related neural activity in the cervical spinal cord in healthy volunteers employing high-resolution functional magnetic resonance imaging (fMRI). We studied histamine-induced itch on the radial forearm and modulated itch intensity by non-noxious cooling. To control for effects of thermal stimulation (i.e., cooling), volunteers also underwent an identical session without histamine. We studied histamine-induced itch on the radial forearm, by using a block design with alternating blocks of non-noxious cooling separated by blocks of skin temperature. Non-noxious cooling of histamine-treated skin compared to cooling of non-treated skin led to a significant increase in itch perception. On the neural level, itch was paralleled by activation in the dorsal horn of the spinal cord at the transition between spinal segment C5 and C6, ipsilateral to the side of stimulation. These results suggest that itch-related neural activity can be assessed noninvasively in humans at the spinal cord. © 2014 Elsevier Inc. All rights reserved.

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The sensation of itch is one of the most frequent complaints in daily clinical routine (Frese et al., 2011). Its sources are manifold and itch can even be induced by verbal suggestion (van Laarhoven et al., 2011). Despite high clinical relevance, the processing of itch in the human CNS is still insufficiently understood. Functional neuroimaging studies employing PET and fMRI revealed a widespread itch-related brain network, comprising the cingulate, parietal, prefrontal, premotor, primary motor, somatosensory and insular cortices (Herde et al., 2007; Hsieh et al., 1994; Mochizuki et al., 2013; Papoiu et al., 2012; Valet et al., 2007). Apart from brain systems, studies in rodents, cats and non-human primates provide increasing evidence that spinal cord processing is substantially involved in pruriception (Akiyama et al., 2009, 2011; Davidson et al., 2009; Kardon et al., 2014; Moser and Giesler, 2013; Nakano et al., 2008; Nishida et al., 2013; Ross et al., 2010; Sun and Chen, 2007; Yao et al., 1992). One example is the inhibition of superficial dorsal horn neurons through scratching (Akiyama et al., 2011; Davidson et al., 2009). Another example in a more clinical context is the well-recognized antagonistic effect of intrathecal opioids — pain-relieving, but itch-inducing (Ballantyne et al., 1988; Ikoma et al., 2003). Especially in itch disorders derived from

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journal homepage: www.elsevier.com/locate/ynimg

⁎ Corresponding author. Fax: +49 40 7410 59955. E-mail addresses: [email protected] (M.F. van de Sand), [email protected] (C. Sprenger), [email protected] (C. Büchel). URL's: http://www.uke.de/institute/systemische-neurowissenschaften/index_ENG.php (M.F. van de Sand), http://www.uke.de/institute/systemische-neurowissenschaften/index_ENG.php (C. Sprenger), http://www.uke.de/institute/systemische-neurowissenschaften/index_ENG.php (C. Büchel).

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spinal cord malfunctions functional BOLD imaging of the spinal cord could become highly beneficial in the future. It is therefore of great interest to investigate itch-related activations non-invasively in the human spinal cord. We targeted spinal pruriception by combining an established itch paradigm in humans (Pfab et al., 2006; Valet et al., 2007) with high resolution spinal fMRI of the cervical spinal cord (Eippert et al., 2009; Geuter and Büchel, 2013; Sprenger et al., 2012). In this paradigm, histamine-induced itch is modulated by non-noxious cooling. Thereby, itch sensations can be increased (25 °C) or decreased (32 °C) in short intervals (Pfab et al., 2006), which is ideally suited for fMRI measurements (Valet et al., 2007). 32 °C is the approximate skin temperature of the forearm (Yosipovitch et al., 1998). Itch was induced applying histamine gel to a peeled skin patch of the radial forearm (Fig. 1); in parallel the intensity of itch sensation was controlled thermally. We recorded itch ratings and BOLD responses in the according spinal segments C5 to C7 in 20 healthy subjects. Employing an additional control session without the application of histamine allowed us to analyze spinal responses to non-noxious cooling and to compare neural activation patterns during itch sensation to phases where itch perception was absent.

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Material and methods

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Subjects

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We invited 23 healthy volunteers aged between 20 and 40 years to participate in the study. Exclusion criteria were MRI-specific exclusion criteria, regular drug intake, pregnancy and lactation, any history of neurological disease, including pain, acute or skin diseases (or a history

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http://dx.doi.org/10.1016/j.neuroimage.2014.12.019 1053-8119/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: van de Sand, M.F., et al., BOLD responses to itch in the human spinal cord, NeuroImage (2014), http://dx.doi.org/ 10.1016/j.neuroimage.2014.12.019

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strong desire to scratch or rasp extensively). Before entering the MRI 119 scanner, subjects were familiarized with the NRS. Ratings involved 120 right hand button presses (index and middle finger). 121

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The experiment consisted of two sessions, in which either histamine or a control solution was applied to a skin patch on the left radial forearm (Fig. 1). Two different temperatures were alternately presented during each session. Neutral temperature blocks (32 °C) reflected the approximate forearm skin temperature (baseline). Non-noxious cooling blocks (25 °C) were used to increase itch sensation under histamine as described previously (Pfab et al., 2006; Valet et al., 2007). As the effect of histamine does not show a determined offset, we had to perform the histamine session after the control session (see below), leading to identical session order in all subjects. Subjects were informed about the purpose of the study, but were blind with respect to the order of treatment (histamine vs control). Before each experiment, a 4 × 5 cm skin patch of the left radial forearm (skin surface of the brachioradialis muscle on the level where the forearm is broadest (in supination); receptive field of the cutaneous antebrachii lateralis nerve) was peeled (EPICONT, GE Medical Systems, Fairfield, CT, USA). The histamine solution consisted of 1% histamine dihydrochloride. 2.5% methyl-cellulose was added to increase viscosity which allowed keeping the solution within the designated skin patch. The control solution was identical, but lacked histamine. Before starting the respective session we allowed each solution to penetrate the skin for 2–3 min. Each session included 18 blocks of non-noxious cooling (20 s) separated by a variable inter-trial interval (duration 21.5 ± 1.5 s). From the 16th second to the end of each block, subjects were asked to rate their perceived itch intensity on a numeric rating scale (NRS) from 0 to 6 where each number was anchored verbally (0: no itch; 1: uncertain/ very mild itch; 2: mild itch, accompanied by the desire to touch or stroke the skin patch; 3: clear itch, accompanied by the desire to scratch; 4: strong itch; 5: very strong itch; 6: unbearable itch, accompanied with a

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All behavioral data were analyzed in Matlab (v7.9, Mathworks, Natick, MA, USA). Itch ratings were averaged for 25 °C- and 32 °C-blocks separately and the difference between both was calculated for each volunteer. The ratings of all four conditions (control_baseline, control_cool, histamine_baseline, histamine_cool) were analyzed using a 2-way repeated measures ANOVA. For all behavioral analyses results were considered significant at p b 0.05.

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of skin disease) and known intolerance to histamine. To avoid extreme differences of spinal segmental size between subjects, only subjects with a body height between 1.6 and 1.9 m were included (mean height ± SD, 1.76 ± 0.08 m). Two volunteers had to be excluded due to an fMRI-related technical problem and one subject showed a disc protrusion. Finally, we were able to analyze data from 7 female and 13 male volunteers (mean age ± SD, 28.2 ± 4.5 years; 19 right-handed). All participants gave their written informed consent and were paid for participation in the study, which was approved by the Ethics Committee of the Board of Physicians in Hamburg and in accordance to the Declaration of Helsinki.

We used Presentation software (Neurobehavioral Systems, Albany, CA, USA) for stimulus presentation, response logging and synchronization with physiological recordings. Thermal stimulation was delivered by a Peltier-Thermode (TSA II, Medoc, Ramat Yishai, Israel) with a 30 × 30 mm surface area. To allow for retrospective physiological noise correction (Brooks et al., 2008; Glover et al., 2000; Piché et al., 2009), which is critical in spinal fMRI, pulse-curve and respiration data were recorded using an MR-compatible physiological monitoring device (Expression; Invivo, Gainesville, USA). Physiological data were digitized using a CED 1401 micro3 and logged on a PC using Spike2 software (both Cambridge Electronic Design, Cambridge, UK). MRI data acquisition was kept similar to previous fMRI studies on pain processing of the human spinal cord (Eippert et al., 2009; Geuter and Büchel, 2013; Sprenger et al., 2012). Subjects were positioned in a 12-channel head coil combined with a 4-channel neck coil with the target region of the spinal cord being centered in the isocenter of the magnet (Magnetom Trio, Siemens, Erlangen, Germany). To avoid movement artifacts the head was stabilized with foamed cushions on both sides. High-resolution (1 × 1 × 1 mm3) T1-weighted anatomical images were acquired using a 3D-MPRAGE sequence (1 × 1 × 1 mm; sagittal slice orientation, repetition time 2.3 s, echo time 3.43 ms, flip angle 9°, inversion time 1.1 s, field-of-view 192 × 240 × 256 mm3) before the first session. The field of view ranged from the body of the corpus callosum to the second thoracic vertebra. For this acquisition, both coils were used, whereas for the acquisition of spinal cord functional images we only used the four channels of the neck coil. Subjects were asked to remain motionless during the entire experiment. Functional T2*-weighted images were acquired using a gradient-echo echoplanar imaging (EPI) sequence (voxel size: 1 × 1 × 5 mm, matrix size: 128 × 128; repetition time: 1170 ms, echo time: 42 ms, flip angle: 70°; GRAPPA with PAT-factor 2). The stack of axial slices was centered on the 4th intervertebral disc, covering the spinal cord from the lower position of the 4th vertebra to the upper part of the 7th vertebra. We acquired 10 slices positioned approximately perpendicular to the spinal cord, using a slice thickness of 5 mm in order to achieve an adequate signal-to-noise ratio despite a high in-plane resolution (1 × 1mm2). To minimize sensitivity to flow effects, flow rephasing in slice direction and spatially-selective saturation pulses superior and inferior to the target volume were used. Furthermore, additional saturation pulses were applied posterior and anterior to the target region, i.e. in the phaseencoding direction, in order to avoid ghosting and minimize inflow artifacts related to pulsatile blood flow in major cervical vessels. To minimize signal intensity variations caused by magnetic susceptibility differences between the intervertebral discs and vertebral bodies for each subject a slice-specific z-shim gradient momentum was used (Finsterbusch et al., 2012). This was determined based on a pre-scan with 21 different gradient moments applied to all slices and by selecting the gradient setting yielding the maximum intensity within the chosen spinal cord region-of-interest. In each session, the first 10 volumes were discarded in order to eliminate T1 saturation effects.

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Fig. 1. Experimental design. A 4 × 5 cm skin patch of the left radial forearm below the crook of the arm was peeled by rubbing with a gauze swab and cream. In sessions one and two this skin patch was treated with control and histamine gel respectively and covered with the thermode head. We waited for 2–3 min to allow the histamine gel to work. For reasons of subject blindness, this was also done for the control gel. Each session included 18 blocks of non-noxious cooling (25 °C, 20 s) separated by 18 jittered skin temperature blocks (32 °C, 21.5 ± 1.5 s). From the 16th second to the end of each single block, subjects had to rate their perceived itch sensation on a numerical rating scale (0–6, no itch to unbearable itch).

MRI data acquisition

Please cite this article as: van de Sand, M.F., et al., BOLD responses to itch in the human spinal cord, NeuroImage (2014), http://dx.doi.org/ 10.1016/j.neuroimage.2014.12.019

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The average itch score for the control session (mean ± SD) was 0.63 ± 0.44 for control_baseline (32 °C) and 1.26 ± 0.95 for control_cool (25 °C) on a numeric rating scale from 0 to 6 (endpoints: no itch, unbearable itch). The average itch score for the histamine session was 1.23 ± 0.88 for histamine_baseline and 2.82 ± 0.85 for histamine_cool (Fig. 2a). Itch-ratings revealed no significant differences between sexes. A 2-way repeated measures ANOVA revealed significant main effects for treatment (F(1,19) = 29.73, p = 2.9 · 10−5) and temperature (F(1,19) = 55.58, p = 4.70 · 10−7) and a significant treatment by temperature interaction (F(1,19) = 21.58, p = 1.76 · 10−4). The differential rating score (cool minus baseline) for the two sessions (mean ± SD) was 1.58 ± 0.77 for the histamine and 0.63 ± 0.85 for the control session (Fig. 2b), indicating that our paradigm successfully generated perceptions which were clearly distinguishable between treatment conditions. As expected, itch ratings were highest for the histamine_cool condition and the “scratch threshold” installed by Pfab et al. (2006) defined as 33% of the maximum was exceeded.

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fMRI results

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Itch-related activations (histamine_cool minus control_cool) 280 In order to estimate itch-related responses in the spinal cord, we in- 281 vestigated activations by comparing non-noxious cooling with and 282

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Preprocessing and statistical analyses of fMRI data were carried out using statistical parametric mapping (SPM8, Wellcome Trust Centre for Neuroimaging, London, UK). Motion correction was performed using a standard rigid body transformation as employed in SPM8 (six degrees of freedom). To minimize the influence of neck and shoulder muscles on the motion correction procedure (Stroman et al., 1999), the cost function was weighted by an automatically created mask (using morphological procedures implemented in Matlab's Image Processing Toolbox) that only included the spinal cord and surrounding tissue (Eippert et al., 2009). To create this mask the area of the CSF is detected (as the variance over functional images is highest (N75%)) and enlarged, leading to a cylindrical binary mask that includes the spinal cord and surrounding area (Inline Supplementary Figure S1). For this mask, regions of high variability (i.e. CSF) were excluded. Masks were specifically created for each subject. Using these masks was required to perform a second realignment of the functional images. We then averaged all functional images and co-registered the T1-weighted to the mean functional image. For co-registration a dilated spinal cord mask (Fig. S1) was applied to the mean functional image. Each subject's anatomical image was then semi-manually co-registered to the masked respective mean functional image using a 6 parameter rigid-body. We used the normalized mutual information, as the objective function. The average distance between sample points was 3 and 1 mm and histogram smoothing was set to 10 × 10. Spatial normalization for group analysis proceeded in the following steps. One T1-weighted anatomical image of a subject with a straight and clearly defined spinal cord was selected from a methodically identical study (Geuter and Büchel, 2013). This image served as the template to which individual anatomical images were then normalized, using linear and nonlinear transformations. To reach the best possible fit of each subjects' image to the template, the template was masked with a cuboid binary mask that included the entire spinal cord (for some exceptions congruency was higher without mask). The success of normalization was checked by eye for each individual subject (for examples see Inline Supplementary Figure S2). Normalization parameters obtained from the anatomical images were then applied to the co-registered functional images and functional images were resampled to a resolution of 1 × 1 × 1 mm. Finally, functional images were smoothed using a 3D isotropic Gaussian kernel with a FWHM of 2 mm. Inline Supplementary Figs. S1 and S2 can be found online at http:// dx.doi.org/10.1016/j.neuroimage.2014.12.019. For the first-level analysis, functional MRI data of 25 °C-blocks and rating intervals were modeled as separate boxcar regressors of the control and histamine sessions, respectively. All regressors were then convolved with the canonical hemodynamic response function. As functional images of the spinal cord are prone to suffer from physiological noise, e.g., resulting from heart beat and respiration (Brooks et al., 2008; Giove et al., 2004; Piché et al., 2009; Stroman, 2005), we corrected not only for noise resulting from cardiac and respiratory sources, but also for fluctuations resulting from the cerebrospinal fluid (CSF). Therefore additional regressors were generated by a selective averaging method described by Deckers et al. (2006) (using 10 bins for cardiac and respiratory effects, respectively). We also corrected for low-frequency noise, e.g., resulting from the CSF (Brooks et al., 2008; Kong et al., 2012). To create this regressor, voxels with a variance in the upper 66.7% percentile of an enlarged mask of the spinal-CSF were included. Furthermore, the 6 motion parameters estimated during previous realignment were included as regressors. In total we included 30 regressors per session: cooling and rating as experimental regressors, 10 cardiac and 10 respiratory, 6 motion, 1 CSF as noise regressors and finally a session constant. Data were high-pass filtered at a cutoff of 128 s. The SPM-default of the intensity-mask-threshold was set to zero. After the first level model estimation we raised the beta images of interest to a group level analysis (ANOVA) and contrasted the histamine

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against the control treatment. Unlike brain imaging, approaches to adjusting p values to control family-wise error rates based on Gaussian random field theory are not suitable for studying extremely small cylindrical structures such as the spinal cord as the smoothness estimation which forms the basis of Gaussian random field correction for multiple comparisons is very conservative and thus leads to increased type II errors. Therefore, uncorrected p values are frequently used when assessing the significance of cord activation (Brooks et al., 2008; Cohen-Adad et al., 2010; Eippert et al., 2009). As performed in previous spinal cord imaging studies, we report an uncorrected statistical threshold of p b 0.005. To solely report spinal responses, we created an inclusive spinal cord mask, using MRIcroN, based on the averaged functional image. Indicated levels of spinal segments are approximations according to spinal nerve rootlet distribution.

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Fig. 2. Itch scores. a) Mean itch scores over all subjects (n = 20) for all 4 conditions on a NRS from 0 to 6 (no itch — unbearable itch). We observed a significant main effect of temperature and treatment. There is a strong interaction effect, resulting in a high itch response for the histamine treatment during non-noxious cooling. b) Differences of itch scores (cool minus baseline) for each treatment. Cooling induced itch is much stronger during histamine compared to control treatment. Error bars reflect standard error of mean. *** p b 0.001.

Please cite this article as: van de Sand, M.F., et al., BOLD responses to itch in the human spinal cord, NeuroImage (2014), http://dx.doi.org/ 10.1016/j.neuroimage.2014.12.019

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In this study we used histamine treatment in combination with nonnoxious cooling to reliably induce the perception of itch during spinal cord fMRI. Corresponding to the stimulation site on the left forearm, we detected itch-related responses in the ipsilateral dorsal horn at the transition between spinal segments C5 and C6 during histamine application compared to control. Importantly, these findings coincide with our behavioral results clearly showing that treatment of a skin patch with histamine in combination with blockwise non-noxious cooling does induce itch in a controllable fashion (Pfab et al., 2006). Furthermore, behavioral data indicates that the itch levels achieved were pronounced as they scored above 33% during the cool condition in the histamine session. Thus, employing the paradigm by Pfab et al. (2006), we robustly modulated itch sensation which is a prerequisite to detect neural responses to itch employing BOLD fMRI. Importantly, this effect cannot be accounted for by expectation, as most of the subjects actually expected that cooling would soothe the itch. The focus of our study was to identify the neural correlates of itch-related processing in the spinal cord. We therefore compared spinal responses during non-noxious cooling in the histamine session – paralleled by maximal itch sensation – to cooling in the control session. Importantly, this comparison controls for the effect of non-noxious cooling. In this analysis, we observed a significantly stronger activation during histamine as compared to control in the dorsal horn of the spinal cord at the transition between segments C5/C6, ipsilateral to the site of stimulation. Histamine-responsive sensory (C−) fibers reveal a very characteristic profile. They are narrowly tuned to histamine, have large innervation territories and very low conduction velocities (Schmelz et al., 1997). Andrew and Craig (2001) exploited this characteristic profile to localize

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the associated projection neurons in the cat spinal cord, which were detected in lamina I. Another study on Fos-like immunoreactivity discovered elevated expression in laminae I and II after histamine treatment (Yao et al., 1992). The response described in our study is located in the dorsal portion of the ipsilateral spinal cord and thus fits to these previous findings. The very lateral location of this response can be explained by the anatomy of the spinal cord in this segment. Cervical segments, especially C6 are transversally expanded and thus more ovally shaped (Kameyama et al., 1996). Fortunately, we were able to rule out strong variation in spinal segment boundaries, as spinal nerve rootlets could be located on the anatomical images. Still, dermatomal overlap is common among mammals (Lee et al., 2008; Sherrington, 1898; Takahashi and Nakajima, 1996) and inter-individual variation of dermatomal innervation might result in higher inter-subject variability. According to these maps responses to stimulation on the radial forearm could emerge in several spinal segments (C5, C6, and C7). We detected a response at the transition between C5 and C6 which might be due to either histamine-responsive fibers from this skin patch only targeting on this level, or elsewise they target C5–C7, but by chance only in C5/C6 the detection threshold was reached. Presumably the truth is somewhere in between, e.g. itchresponsive sensory neurons of the radial forearm are predominantly targeting the intersection of spinal segments C5 and C6. Our data suggest that this histamine-induced response is related to itch, as behaviorally itch was highest during histamine_cool and as its axial position is in line with previous research on histamineresponsive neurons. Subsequently this paradigm allowed us to successfully modulate histamine-induced itch sensation and to record its spinal correlate. Thus temperature seems to influence itch processing already in the range of early sensory processing (sensory, inter- and/or projection neurons). The mechanisms of thermally controlled itch are only partially understood yet. Current concepts regarding the underlying mechanisms range from an interaction on the receptor level (Bíró et al., 2007), temporal disinhibition of pruriceptive spinal neurons (McCoy et al., 2013) to insular regulation (discussed in Valet et al. (2007)). There is increasing evidence that thermosensitive transient receptor potential (TRP-) channels play a major role in thermal itch modulation. First, thermosensitive TRP-channels are more likely to become activated in a certain range of temperature (Voets et al., 2004). Second, TRPV1

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without histamine (histamine_cool minus control_cool; main contrasts in Supplementary material 3). Behaviorally, non-noxious cooling on histamine-treated skin led to a significant increase in perceived itch (see blue bars in Fig. 2a). Thus, this analysis (i) parallels the behavioral analysis (cf. Figs. 2b & 3c), (ii) controls for cooling and (iii) tests for itch-related activations in the spinal cord. This contrast revealed a response in the dorsal horn of the spinal cord (t(19) = 3.03, p = 0.003, Figs. 3a & b), in the caudal C5 to cranial C6 spinal segment.

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Fig. 3. Itch-related response in the spinal cord (histamine_cool minus control_cool). a), b) The effect related to itch (histamine_cool N control_cool) is overlaid on a sagittal slice of the T1weighted template. The contrast reveals a histamine-induced signal increase, attributable to the ipsilateral dorsal horn at the transition between spinal segments C5 and C6. The activation in lower a) is most likely CSF-related noise; no spinal mask was used. Ten slices were recorded between green dashed lines. The light red line indicates the axial slice (b)), where results are overlaid on the mean functional image; v: ventral, d: dorsal, l: left, r: right. c) Parameter estimates for the itch stimulation extracted from the peak voxel. Parameter estimates were higher for the histamine treatment compared to control (t(19) = 3.03; p = 0.003; the color bar indicates t-values and the visualization threshold is set to p b 0.01. Error bars represent SEM).

Please cite this article as: van de Sand, M.F., et al., BOLD responses to itch in the human spinal cord, NeuroImage (2014), http://dx.doi.org/ 10.1016/j.neuroimage.2014.12.019

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Authors' contribution

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MvdS, CS & CB designed the experiment. MvdS conducted the experiments. MvdS & CS analyzed the data. MvdS, CS & CB wrote the paper.

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We thank Stephan Geuter for his support at several steps of the study, Jürgen Finsterbusch for the configuration of the MR pulse sequences and Simon Schede for his support on Fig. 1. The current work was supported by the European Research Council (ERC), ERC-2010AdG_20100407.

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The authors declare no competing financial interests.

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Taken together, our results provide evidence for the feasibility of detecting itch responses non-invasively in the human spinal cord using BOLD fMRI. These data represent the basis for subsequent investigations of itch-related diseases that include itch as a major symptom. Finally fMRI of the spinal cord could be a valuable method to bridge the gap between peripheral microneurography and brain imaging and to address interactions of itch with other modalities, such as pain.

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(vanilloid-1) is additionally involved in histamine responses in mice (Imamachi et al., 2009). Further regulatory mechanisms, also on higher neural levels might come to play to cause a cooling-induced itch response. When investigating cerebral effects using the same paradigm, no significant changes were detected to non-noxious cooling in the control session (Pfab et al., 2010; Valet et al., 2007), indicating that the extent of cooling itself is very low. In contrast, several increases (presupplementary motor area, dorsolateral prefrontal cortex, anterior insula and inferior parietal cortex) and decreases (supplementary motor area, medial frontal, orbitofrontal, dorsal anterior cingulate, primary motor and primary somatosensory cortex) of activity to cooling during the histamine session were reported (Valet et al., 2007). The applied thermal stimulation protocol does not allow for investigation of purely histamine-specific responses, but rather represents the interaction of histamine and thermal stimulation. However, behaviorally the difference between cooling combined with histamine and cooling alone gives rise to pronounced differences in itch perception. Therefore comparing these conditions best captures itch-related processing.

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