Bodily Sensory Inputs and Anomalous Bodily Experiences in Complex Regional Pain Syndrome: Evaluation of the Potential Effects of Sound Feedback

Neuroscientific studies have shown that human's mental body representations are not fixed but are constantly updated through sensory feedback, including sound feedback. This suggests potential new therapeutic sensory approaches for patients experiencing body-perception disturbances (BPD). BPD can occur in association with chronic pain, for example in Complex Regional Pain Syndrome (CRPS). BPD often impacts on emotional, social, and motor functioning. Here we present the results from a proof-of-principle pilot study investigating the potential value of using sound feedback for altering BPD and its related emotional state and motor behavior in those with CRPS. We build on previous findings that real-time alteration of the sounds produced by walking can alter healthy people's perception of their own body size, while also resulting in more active gait patterns and a more positive emotional state. In the present study we quantified the emotional state, BPD, pain levels and gait of twelve people with CRPS Type 1, who were exposed to real-time alteration of their walking sounds. Results confirm previous reports of the complexity of the BPD linked to CRPS, as participants could be classified into four BPD subgroups according to how they mentally visualize their body. Further, results suggest that sound feedback may affect the perceived size of the CRPS affected limb and the pain experienced, but that the effects may differ according to the type of BPD. Sound feedback affected CRPS descriptors and other bodily feelings and emotions including feelings of emotional dominance, limb detachment, position awareness, attention and negative feelings toward the limb. Gait also varied with sound feedback, affecting the foot contact time with the ground in a way consistent with experienced changes in body weight. Although, findings from this small pilot study should be interpreted with caution, they suggest potential applications for regenerating BDP and its related bodily feelings in a clinical setting for patients with chronic pain and BPD

Additional file 1: Tables S1–S5. of Prolonged transfer of feces from the lean mice modulates gut microbiota in obese mice

Table S1. Taxonomic Assignments Table. Full taxonomic assignments in SILVA Taxonomy, as classified by Mothur. Taxa are given with bootstrap values for each taxonomic level. Taxa are classified to family, unless there was only one classifiable genus (or lower level) present in the family. Table S2. Serum biochemical parameters at the end of the experiment. Normal diet (ND), high-fat diet (HFD), HFD supplemented with feces of ND-fed mice (HFDS). Table S3. Differential taxa abundances between weeks 0 and 12 and weeks 0 and 28, tested with paired Mann-Whitney U-tests for each experimental group. Taxon = taxon denotation (full taxonomic assignments in SILVA taxonomy are presented in Additional file 1: Table S1); Mann-Whitney U statistic = statistic from paired Mann-Whitney U-tests; pValue = p-value from paired Mann-Whitney U-test; qValue = p-value after FDR correction. Table S4. Results of model comparison for taxa. Taxon = taxon denotation (full taxonomic assignments in SILVA taxonomy are presented in Additional file 1: Table S1); pValue time and pValue diet = p-values for comparisons between null models, models including time and models including time and diet, respectively; qValue time and diet = p-values after FDR correction. Table S5. Differential taxa abundances at 12 and 28 weeks. Taxon = bacteria taxon in SILVA taxonomy, genera (if applicable) are given in parentheses; week = week of experiment; comparison = groups compared; mean 1 group = mean abundance of taxon in first group in comparison field; mean 2 group = mean abundance of taxon in second group; pValue = p-value of Mann-Whitney U-tests; qValue = p-value after FDR correction. Taxa that were differentiated in two groups at both time points are highlighted in bold text. (XLSX 60 kb

The <i>N</i>-Reductive System Composed of Mitochondrial Amidoxime Reducing Component (mARC), Cytochrome b5 (CYB5B) and Cytochrome b5 Reductase (CYB5R) Is Regulated by Fasting and High Fat Diet in Mice

<div><p>The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing <i>N</i>-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in <i>N</i>-reductive drug metabolism such as the activation of amidoxime prodrugs. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an <i>N-</i>reductive biotransformation assay to gain activity data. Indeed all proteins of the <i>N</i>-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation <i>in vivo</i>, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the <i>N</i>-reductive reaction, benzamidine, was determined. Albeit altered <i>in vitro</i> activity, no changes in the metabolite concentration <i>in vivo</i> were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased <i>N</i>-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism.</p></div

Additional file 2: Figure S1. of Prolonged transfer of feces from the lean mice modulates gut microbiota in obese mice

Rarefraction curves from all samples on family level drawn with MEGAN5 software. (PDF 4369 kb

Primers used for expression studies.

<p>Primers used for expression studies.</p

Glucose-dependent changes with expression, abundance and activity of the <i>N</i>-reductive system in HepG2 and Hepa 1.6 cell lines.

<p><b>A</b> Expressions of mARC2, mARC1, CYB5R and CYB5B in HepG2 and Hepa 1.6 cell lines cultured without or with 1.0/4.5 g/l glucose determined by qRT-PCR. Statistical significance was assessed by the U-test. <i>p</i>-values <0.05 were considered significant (*); (**) <i>p</i>-value <0.001, ns = not significant. <b>B</b> Protein levels of mARC2, mARC1, CYB5R and CYB5B in hepatoma cell lines cultured without or with 1.0/4.5 g/l glucose, examined by Western Blot. R = recombinant proteins/control <b>C </b><i>N</i>-reductive activity determined in in hepatoma cell lines by determining the reduction of model compound benzamidoxime. The resulting metabolite benzamidine was quantified by HPLC analysis. Determined activities are means ± SD of six biological samples, each measured as duplicates. Statistical significance was assessed by the U-test. <i>p</i>-values <0.05 were considered significant (*); (**) <i>p</i>-value <0.001.</p

HFD but not hyperphagia increases mARC abundance and <i>N</i>-reductive activity in mice.

<p>Both a group of C57BL/6W and ob/ob-mice were fed with regular diet, another group of C57BL/6W mice was fed with HFD. Mice were sacrificed and livers collected. <b>A</b> Expressions of mARC1, mARC2, CYB5R and CYB5B determined with qRT-PCR, normalized on expression of Mcoln1 (Mucolipin-1) and Hmbs (hydroxymethylbilane synthase). Expression shown as means ± SD of 6 biological samples. Statistical significance was assessed by the U-test. p-values <0.05 were considered significant (*). <b>B</b> Protein levels of mARC1, mARC2, CYB5B and CYB5R in liver homogenates examined by Western Blot. <b>C </b><i>N</i>-reductive activity determined by the reduction of model compound benzamidoxime in liver homogenate. The resulting metabolite benzamidine was quantified by HPLC analysis. Determined activities are means ± SD of four biological samples, each measured as duplicates. Statistical significance was assessed by the U-test. p-values <0.05 were considered significant (*); (***) p-value <0.0001; ns = not significant.</p

Scheme of the <i>N</i>-reductive reaction with model compound benzamidoxime by mARC, CYB5B and CYB5R.

<p>Electrons are transferred from NADH via FADH in CYB5R, hem in CYB5B and Moco in mARC1/2 to benzamidoxime, the latter is reduced to benzamidine.</p

24 h fasting decreases mARC protein levels and <i>N</i>-reductive activity in mice.

<p>Two groups of 14 C57BL/6W mice were fed with regular diet, one group were food deprived for 24 h (fasted) before sacrifice and liver collection, the second had full access to food and water (non-fasted). <b>A</b> Expressions of mARC1 and mARC2, determined with qRT-PCR, normalized on expression of Mcoln1 (Mucolipin-1) and Hmbs (hydroxymethylbilane synthase). Statistical significance was assessed by the U-test. <i>p</i>-values <0.05 were considered significant (*); (**) <i>p</i>-value <0.001; (***) <i>p</i>-value <0.0001. <b>B</b> Protein levels of mARC1, mARC2 and histone H3 (loading control) examined by Western Blot. Each sample consisted of equal protein amount of two individuals. <b>C </b><i>N</i>-reductive activity determined by the reduction of model compound benzamidoxime in liver homogenate. The resulting metabolite benzamidine was quantified by HPLC analysis. Determined activities are means ± SD of 14 biological samples, each measured as duplicates. Statistical significance was assessed by the U-test. <i>p</i>-values <0.05 were considered significant (*); (***) <i>p</i>-value <0.0001.</p

Boxplot of community α diversity at day 0 (prior to supplementation) according to the Simpson index and categorized according to gestational age at birth.

<p>Data are shown only for the weeks in which at least five infants were born.</p