With positron emission tomography (PET) it has become possible to image this phenomenon in vivo and over the last years patterns of microglia activation corresponding with the known distribution of neuropathological changes in these disorders have been demonstrated using this technique. In addition the effects of interventions aimed at suppressing microglia activation as part of interventional trials have successfully been demonstrated. Current research aims at evaluating PET tracers for microglial activation with more favorable properties than the prototypical [ Inflammation is a reaction of living tissues to injury and acute and chronic inflammation can be distinguished. Acute inflammation comprises the immediate and early response to an injurious agent and is basically a defensive response that paves the way for repair of the damaged site. Chronic inflammation results from stimuli that are persistent. In the periphery, inflammation consists of leukocytic infiltrates characterized by polymorphonuclear cells (neutrophils) and mononuclear cells .Over the last years, the term “neuroinflammation” has come into widespread use. This term generally denotes chronic, CNS-specific, inflammation-like glial responses that do not reproduce the classic characteristics of inflammation in the periphery but that may cause and/or sustain neurodegenerative events including plaque formation, dystrophic neurite growth, and excessive tau phosphorylation . NeuroinMicroglia are normally quiescent, brain macrophages and represent the resident immuno-competent cells of the central nervous system. Recent fate mapping studies have shown that under your homeostatic conditions microglia are not derived from the bone marrow but from haematopoietic stem cells in the yolk sac during embryogenesis . Del RioThis morphological change has been well documented in the facial nerve transection model, where microglial activation can be assessed in an environment without blood–brain barrier disruption or migration of new bone marrow-derived macrophages. The model demonstrates the capacity of resident microglia to undergo morphological changes and activation with expression of new surface markers and to proliferate around motor neurons of the facial nucleus .Microglia activation in vivo occurs as a graded response. The transformation of microglia into potentially cytotoxic cells is under strict control, mainly in response to neuronal or terminal degeneration, or both. Activated microglia are mainly scavenger cells but also perform various other functions in tissue repair and neural regeneration . They foThe main pathological finding in IPD (idiopathic Parkinson’s disease) is the degeneration of dopaminergic projections from the substantia nigra pars compacta to the caudate nucleus and putamen (striatum). Intraneuronal Lewy bodies and Lewy neurites composed of the protein α-synuclein are the pathological hallmarks of the disease. However, the neuropathological changes are not confined to the substantia nigra . This isIn idiopathic Parkinson’s disease activated microglia have initially been described in the substantia nigra  and moreThe exact contribution of microglial activation to the degenerative process in IPD is presently not well understood. There is evidence from humans  and monkIn multiple system atrophy (MSA) typical histopathological changes consist of neuronal loss in the nigrostriatal and olivopontocerebellar pathways with α-synuclein-positive, argyrophilic staining, glial cytoplasmic inclusions associated with reactive astrocytes and activated microglia –20 .In PSP  there is atrophy of the brainstem, internal globus pallidus, amygdala, frontal and parietal lobe, and nigral depigmentation. Microscopically, neuronal loss is found in brainstem oculomotor nuclei, pallidum, substantia nigra, subthalamic nucleus, and frontal cortex. The pathological hallmarks of PSP are intraneuronal neurofibrillary tangles (NFTs) composed of abnormally phosphorylated microtubule-associated tau-protein and neuropil threads . The celGross pathological findings in CBD  include narrowing of cortical gyri often in a peri-Rolandic distribution. The superior frontal gyrus and inferior parietal area are usually most affected while the inferior frontal gyri, temporal, and occipital lobes are spared. The atrophy is characteristically asymmetric  and detaAs shown in these studies referenced above, the neuropathology of parkinsonian disorders is closely associated with microglial activation. The exact role of neuroinflammation in neurodegenerative disorders, however, is unclear. Post-mortem studies and animal experiments have numerous methodological limitations; for a review see .In vivo imaging in man using positron emission tomography (PET) can overcome some of these, and the principles and applications are detailed below.One of the molecules expressed during microglial activation is the mitochondrial translocator protein 18 kDa (TSPO), formerly known as the ‘peripheral benzodiazepine binding site’ (PBBS) , 26. ThiN-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide), a highly specific TSPO ligand, exhibits low binding in the normal brain, while increased binding is indicative of increased TSPO expression in microglia following neuronal injury [11C-labeled (R)-isomer of PK11195 can thus be used to image activated microglia in vivo. While [11C]-(R)-PK11195 is the best pre-clinically and clinically characterized PET ligand to image microglial activation, new, often [18F]-based, radiotracers for microglial activation have been developed recently, but so far have only been evaluated to a limited extent in a clinical context -DOPA PET. Eight of the patients were followed up longitudinally over 24 months, and, although the disease had clinically progressed, there was no significant change in the extent of microglial activation. In contrast, Ouchi and colleagues [11C]-(R)-PK11195 BP in the midbrain, which was negatively correlated with dopamine transporter binding measured by [11C]-CFT PET and positively correlated with motor severity. Differences in methodology as well as motor severity (mean UPDRS motor score 32.0 compared to 10.4) may explain the differences in distribution of [11C]-(R)-PK11195 binding and other findings between these two studies.In Parkinson’s disease increased [controls  Fig. 2)11C]-(R)-lleagues  studied 11C]-(R)-PK11195 binding as by Gerhard et al. This study also included patients with dementia with Lewy bodies, in whom widespread cortical microglial activation was seen in addition to subcortical changes -FEPPA -FEPPA . However11C]-(R)-PK11195 binding was conducted in five patients with multiple system atrophy (MSA) compared to controls -(R)-PK11195 binding, but no clinical benefit was shown in the overall study -(R)-11C]-PBR28 PET the authors reported a reduction of distribution volume of the radioligand after 4 and 8 weeks of treatment compared to the placebo group where no change was found.Recently one of the new TSPO PET ligands has been used to measure microglial activation in Parkinson’s disease patients treated with AZD3241, a selective and irreversible inhibitor of myeloperoxidase with the putative mode of action of a reduction of sustained neuroinflammation through reduced oxidative stress . Using [11C]-PBR28 binding in healthy controls and patients with Parkinson’s disease and it is therefore currently unknown whether 11C-PBR28 increases in PD patients follow a similar pattern as in studies with [11C]-(R)-PK11195.So far, however, there is no published study comparing [11C]-(R)-PK11195 in parkinsonian disorders with tau-pathology were also able to show prominent microglial activation in vivo. In progressive supranuclear palsy, elevated [11C]-(R)-PK11195 BP was observed in the caudate, putamen, pallidum, substantia nigra, midbrain, thalamus, cerebellum and frontal lobe [PET studies using[tal lobe . In the 11C]-(R)-PK11195 BP in caudate, putamen, substantia nigra and frontoparietal cortex -(R)-PK11195 BP in patients with MSA-P and PSP with MR markers of tissue damage such as apparent diffusion coefficient was investigated; here, microglial activation did not correlate with putaminal water diffusivity.In a recent study  the relaOver the last few years targets involved in the neuroinflammatory process in neurodegenerative disease other than the TSPO have been identified that are currently evaluated using PET ligands.2R), which is involved in peripheral immune system function, and is also upregulated in CNS disorders showing microglial activation [11C]-NE40, which shows binding to the human CB2R overexpressed in rodent striatum in preclinical PET studies [2R PET studies in PD have been published.One very interesting target is the cannabinoid receptor type 2  demonstrated specific binding to cortical areas with large numbers of reactive astrocytes and microglia [11C]-deuteriodeprenyl (DED) has been utilized as a PET tracer due to its kinetic advantages, including increased sensitivity -DED binding was highest in MCI patients compared to healthy controls and AD patients overall. PiB-(a PET marker for amyloid) positive MCI patients had the highest levels of DED binding while AD patients had the least [3H]-PiB, [3H]-PK11195 and [3H]-L-deprenyl in AD brain tissue [11C]-DED on PET was suggestive of astrocytosis -(R)-PK11195 PET is an imaging modality with the ability to demonstrate one aspect of the neuropathology in neurodegenerative disorders in vivo and longitudinally in a minimally invasive fashion.-(R)-PK11195 PET data in neurodegenerative disease [11C]-labeled tracer [11C]-(R)-PK11195 has only a short half-life and necessitates a cyclotron on site for tracer production, which limits wider clinical application.The optimal method of analysis for PET data is also the subject of debate, as the selection of an anatomically defined reference region due to constitutive expression of TSPO within the brain and potentially widespread pathology in neurodegenerative disorders, is difficult. Methods using kinetic analysis to identify a reference tissue cluster have, therefore, been employed in the analysis of -(R)-PK11195 has further complicated the analysis of these data. Thus, studies utilizing such ligands require analysis of TSPO binding affinity to correct for this factor.Currently significant effort goes into the development of novel TSPO ligands for PET imaging in neurodegenerative diseases. Additionally, the recent finding of populations of high- and low-affinity binders for TSPO ligands other than [11C]-(R)-PK11195 will show whether any of the new putative TSPO tracers are indeed superior in the clinical context. As there are known species differences in TSPO binding, in vitro and preclinical animal data do not always directly translate into clinical applications in humans.Only further clinical evaluations and direct comparisons with [


Gilles de la Tourette syndrome is a neuropsychiatric disorder characterized by motor and phonic tics that can be considered motor responses to preceding inner urges. It has been shown that Tourette patients have inferior performance in some motor learning tasks and reduced synaptic plasticity induced by transcranial magnetic stimulation. However, it has not been investigated whether altered synaptic plasticity is directly linked to impaired motor skill acquisition in Tourette patients. In this study, cortical plasticity was assessed by measuring motor-evoked potentials before and after paired associative stimulation in 14 Tourette patients  and 15 healthy controls . Tic and urge severity were assessed using the Yale Global Tic Severity Scale and the Premonitory Urges for Tics Scale. Motor learning was assessed 45 minutes after inducing synaptic plasticity and 9 months later, using the rotary pursuit task. On average, long-term potentiation-like effects in response to the paired associative stimulation were present in healthy controls but not in patients. In Tourette patients, long-term potentiation-like effects were associated with more and long-term depression-like effects with less severe urges and tics. While motor learning did not differ between patients and healthy controls 45 minutes after inducing synaptic plasticity, the learning curve of the healthy controls started at a significantly higher level than the Tourette patients' 9 months later. Induced synaptic plasticity correlated positively with motor skills in healthy controls 9 months later. The present study confirms previously found long-term improvement in motor performance after paired associative stimulation in healthy controls but not in Tourette patients. Tourette patients did not show long-term potentiation in response to PAS and also showed reduced levels of motor skill consolidation after 9 months compared to healthy controls. Moreover, synaptic plasticity appears to be related to symptom severity. Gilles de la Tourette syndrome (GTS) is a neuropsychiatric disorder characterized by motor and phonic tics that typically emerge during early childhood N-methyl-D-aspartate (NMDA) receptor gated channels Synaptic plasticity refers to the capacity of nerve cells to alter their structural and functional properties such as strengthening of a synapse by long-term potentiation (LTP). LTP is defined as an activity dependent long lasting enhancement of synaptic transmission, while long-term depression (LTD) refers to a long lasting attenuation of synaptic transmission; both LTP and LTD are referred to as synaptic plasticity and are thought to constitute the neuronal basis for learning and memory LTP – and LTD-like neuroplasticity can be induced in the primary motor cortex (M1) in humans using techniques such as repetitive TMS protocols including theta burst stimulation (TBS), high frequency stimulation (HFS) and paired associative stimulation (PAS). It has been shown that PAS induces synaptic plasticity more effectively than TBS, at least in healthy participants 25), corticospinal excitability increases 10), excitability decreases N20).In the PAS protocol, an electrical, peripheral stimulus is applied to the wrist, before a TMS stimulus is delivered to the contralateral M1. The PAS protocol displays properties that are also associated with synaptic plasticity induced in single cells. First of all, if the peripheral, afferent stimulus arrives at the same time or shortly before the TMS stimulus in M1  are commonly measured as the dependent variable in TMS paradigms inducing synaptic plasticity because they reflect corticospinal excitability. Studies in healthy participants show an increase in MEP amplitudes after PASPrevious studies employing TBS and HFS have found reduced synaptic plasticity in GTS patients as compared to healthy controls 25-induced LTP and motor learning in the rotary pursuit task has been further demonstrated in a sample of healthy controls and schizophrenia patients In order to relate motor skill acquisition and consolidation to synaptic plasticity in GTS patients, we investigated whether synaptic plasticity in M1 is impaired in uncomplicated GTS patients, employing the adapted PAS protocol as described by Ziemann and colleagues (2004) N20 were determined on the basis of MEP amplitudes and cortico-spinal excitability measured by input-output (IO) curve changes Resulting effects of PASThe aim of this study was to corroborate findings of altered synaptic plasticity in uncomplicated GTS patients and relate synaptic plasticity directly to the ability to acquire and consolidate motor skills. Our results confirm that synaptic plasticity was altered in GTS patients as compared to healthy controls. While motor skill acquisition was normal in GTS patients, motor skill consolidation was impaired and only correlated with induced LTP in healthy controls but not in patients.Fourteen patients  with a diagnosis of GTS according to DSM-IV-TR criteria were recruited from the University hospital Hamburg-Eppendorf in Hamburg. Patients fulfilling criteria for OCD according to the structured clinical interview for DSM-IV Axis I disorders (SCID-I), ADHD according to DSM-IV-TR or other neurological or psychiatric comorbidities were excluded from the study. Thus, all patients had uncomplicated GTS exhibiting no clinically relevant comorbidities. GTS symptom severity within a week before the study was assessed by a clinician using the Yale Global Tic Severity Scale (YGTSS) Fifteen healthy age-matched individuals  without a history of psychiatric disorders or neurological diseases were recruited as a control group. All participants were tested between 1–7 pm to avoid confounding effects of circadian rhythm. All participants were right-handed as assessed by the Edinburgh handedness Inventory N20 protocol was administered. MEPs were measured before PASN20, immediately after PASN20 and 30 min after PASN20. Participants were given a 10 min break and were then asked to complete 12 trials of the rotary pursuit task, overall 45 min after administration of the PASN20 protocol. Additionally, all participants were invited for a second testing session of the rotary pursuit task 9 months later. Of the 29 participants, 10 patients and 12 healthy controls were able to attend the second testing session.Before the start of the experiment, all participants completed a TMS safety screening. None of the participants had a family history of epilepsy or had undergone neurosurgery. Thereafter, the PASParticipants were seated in a comfortable reclining chair with their hands resting on a table and were asked to relax and keep their eyes open. To insure that all participants stayed alert during the whole TMS procedure, a standardized attention test was administered The optimal location for the magnetic coil was defined as the site where the largest MEPs in the right abductor pollicis brevis (APB) muscle could be produced by slightly suprathreshold stimulation of the contralateral M1. The location and orientation of the coil was then marked on the scalp with a soft pen. Next, the resting motor threshold was determined as the lowest stimulus intensity capable of inducing peak-to-peak MEPs with amplitudes of more than 50 µV in the relaxed APB in at least 5 out of 10 consecutive trials. TMS stimulus intensities are generally reported as percentage of maximum stimulator output (100%). The test stimulus intensities applied during all following stimulations were adjusted to evoke peak-to-peak MEP amplitudes of approximately 1 mV in each participant. The sensory perception threshold for peripheral stimulation was defined as the least intense electrical stimulus that could be perceived by each participant and was assessed by increasing and decreasing stimulus intensity 10 times around the first noticeable stimulus.Somatosensory evoked potentials were then obtained from each participant to assess how long it takes for an electrical stimulus to travel from the median nerve at the wrist to the cortex. For this purpose, 300 electrical stimuli  were applied to the right median nerve and the average response time was measured over sensory motor cortex . For reliability purposes, somatosensory evoked potentials were measured twice and results were averaged. Based on this method, the interval between the electrical stimulus applied to the wrist and the magnetic stimulus applied to the cortex in the PAS protocol can be individualised and thus optimised for each participant.N20 consisted of 225 pairs of single, peripheral electrical stimuli at the median nerve (300% of the sensory perception threshold) and suprathreshold TMS over the hand area of the contralateral M1 (adapted from N20 (T1) , immediately after PASN20 (T2) and 30 min later (T3) . The measured output slope of IO curves is sensitive to the order in which the stimulus intensities are applied (ascending or descending), hence five single pulses per intensity were delivered twice in an ascending order of stimulus intensity.Surface electromyography (EMG) recordings were made with silver surface electrodes placed over the right APB using a tendon-belly montage. EMG signals were amplified and filtered (20 Hz to 1 kHz). The signals were sampled at 5000 Hz and digitized using an analogue-digital converter , Cambridge, UK). Off-line data analysis was performed with the Signal software . Auditory feedback on muscle relaxation was provided through a loudspeaker connected to the EMG channel.All TMS measurements were performed with a Magstim 200 magnetic stimulator . A figure-of-eight coil with an outer diameter of 70 mm (Magstim Company) was held tangentially over the scalp at a 45° angle to the sagittal plane with a coil orientation inducing posterior-anterior currents in the brain.N20 protocol was applied over the median nerve at the wrist with a standard stimulation block  at a stimulation width of 200 µs and a duration of 1 ms.Electrical stimulation for somatosensory evoked potentials measurements and during the administration of the PASThe motor skill task consisted of a computerized version of the rotary pursuit task MEP amplitudes were measured semi-automatically peak-to-peak for each frame using Signal software (customized script). Mean values were calculated for each participant by averaging the MEP amplitudes, excluding single trials that deviated more than 2.5 SDs from the mean. The data pre-processing was also conducted by script. Subsequent off-line data analysis in SPSS was not conducted blind to the diagnosis.N20. In case of a violation of the sphericity assumption, the Greenhouse-Geisser correction was chosen. Post-hoc tests, if applicable, were conducted using independent samples t-tests and paired-samples t-tests. Results were considered significant if p<0.05. An “MEP change” variable was calculated by subtracting mean MEP amplitude values at T2 from values at T1 so that positive values represent LTP-like changes and negative values represent LTD-like changes. In addition, to determine synaptic plasticity independent of the direction (LTP or LTD), absolute values of MEP changes from time 1 to time 2 (|MEP T2-T1|) were compared between the groups by an independent samples t-test.Repeated measures analyses of variance (ANOVA) were carried out with time  as a within-subjects factor and group as a between-subjects factor to detect differences in mean MEP amplitude in response to PASN20 effects on IO curves, 3×5 repeated measures ANOVAs with time  and stimulus intensity  as within-subjects factors were carried out for both groups. Slopes of each curve were assessed for each participant by fitting the data to a linear regression function. The slope values were then entered into a repeated measures ANOVA with time  as a within subjects factor and group as a between-subjects factor. Hypothesis-driven correlations were performed in patients between clinical scores, MEP change, IO curve slopes, resting motor threshold and strength of test stimulus.To evaluate PASTo investigate differences in motor learning between the groups, a repeated measures ANOVA was carried out with trial (1–12) and time  as within-subjects factors and group as a between subjects factor. Further, MEP change was correlated with motor performance. Two healthy controls had 4 missing data points at rotary pursuit 1. The missing data points were replaced by the mean values of all other healthy controls for the respective trial. For correlation analyses, the 4 trials of the 3 blocks were averaged for time 1 and 2 respectively.N20 . MEPs before and after PASN20 were obtained from 14 GTS patients and 15 healthy controls. IO curve data were not available in one GTS patient because this patient experienced higher stimulation intensities as uncomfortable. Groups did not differ with respect to gender or mean age (see r = .08) nor with MEP change from T1 to T2 (r = −.07).Healthy controls and GTS patients performed equally well in the attention test administered during PAS age see . Also, m age see . Age wasF  = 4.79, p = .012]. Post-hoc t-tests showed that MEP amplitudes at baseline did not differ between groups . MEP amplitudes increased from T1 to T2  and decreased from T2 to T3 in healthy controls . In contrast, there was no mean MEP amplitude difference between T1 and T2 , or between T2 and T3  in GTS patients (see t(9)  = 1.16, p = .278] and T2 to T3 ). T1 and T3 did not differ significantly from each other in either group, indicating that the PASN20 effect had worn off after 30 min  with „group“ as a between-subjects factor (N = 29) revealed a significant interaction between time and group . An explorative ANOVA was then conducted for each group separately to see whether there was a detectable effect of PASN20 in one of the groups.Concerning the IO curve data, the 3×5 repeated measures ANOVA („time“ x „stimulus intensity”) with “group” as a between-subjects factor did not reveal any significant interactions involving “group”. As expected, there was a significant main effect for stimulus intensity  and a marginally significant effect for time . Explorative post-hoc t-tests showed that overall mean MEP amplitudes at T2 were significantly larger than those at T1  indicating a general increase in cortico-spinal excitability after PASN20 in the control group, corroborating the results from the main experiment   = 22.76, p<001] indicating a normal ascending response of MEPs to higher intensities. There was no PASN20 effect in GTS patients (see The 3×5 repeated measures ANOVA („time“ x „stimulus intensity”) revealed no significant interaction of time and stimulus intensities for IO curve data in controls. As expected, there was a significant main effect of stimulus intensity with higher intensities eliciting higher MEP amplitudes , indicating that overall, plasticity effects were comparable. However, more GTS patients than healthy controls (57% compared to 33%) showed an LTD-like change in response to PASN20.The amount of MEP change following PASr = .56, p = .036), i.e. higher tic severity was associated with lower cortical excitability suggesting a generally decreased resting motor excitability in severely affected GTS patients. Moreover, total tic severity correlated positively with MEP change from T1 to T2 , indicating LTP-like plasticity in more severely affected patients and LTD-like plasticity changes in patients with fewer tics . Correlations between the resting motor threshold, and MEP change with clinical measures are reported in The mean resting motor threshold did not differ between the groups. However, variability was higher in GTS patients  than in healthy controls . YGTSS scores  correlated positively with resting motor threshold . Moreover, the strength of premonitory urges was highly correlated with MEP change  indicating that patients with higher LTP-like changes had stronger premonitory urges while patients with stronger LTD-like responses reported fewer or less severe urges  or previous medication Information about premonitory urges was obtained from 12 patients. Premonitory urges and tic severity were positively associated   = 69,75, p<001]. The assumption of sphericity was violated for the within subjects tests, therefore the results of the multivariate tests will be reported. The only significant result apart from the linear learning across trials was a significant three-way interaction between learning curve, time and group . Post hoc t-tests revealed that while there was no difference between patients and healthy controls in the first trial of the rotary pursuit task at time 1 , healthy controls started their second learning curve (time 2) at a significantly higher level than GTS patients   at time 1 and 2 that can be explained by variance in synaptic plasticity. In healthy controls, 7% of the variance in motor learning in the rotary pursuit task 1 can be explained by PASN20 related synaptic plasticity. In contrast, 31% of the variance in motor learning in the rotary pursuit task 2 can be explained by synaptic plasticity induced 9 months earlier. For GTS patients, 33% of the variance in the rotary pursuit task 1 and 26% of the variance in the rotary pursuit task 2 can be accounted for by changes in synaptic plasticity induced by PASN20. This measure does not reflect causality but merely assesses the size of the association without taking sample size into account. According to these results, there is a difference in the association between motor learning and synaptic plasticity between time 1 and 2 in GTS patients but it is not as clear as in healthy controls.Mean learning in block 1 and 3 at time 1 was significantly negatively correlated with MEP change in GTS patients [k 3] see , whereas.19] see . In heal.03] see . Amplitu.23] see . The sizPerformance in the rotary pursuit task 1 and the rotary pursuit task 2 did not correlate significantly with the total YGTSS score or premonitory urges.N20 increased in healthy controls N20 that was correlated with less severe urges and fewer tics. Both groups performed equally well in the motor task immediately following PASN20. However, healthy controls performed significantly better than GTS patients in the first trial of the motor task 9 months later, indicating that long-term consolidation processes differed between the two groups.The main finding of this study is that M1 synaptic plasticity in adults with uncomplicated GTS differs from healthy controls. As expected, mean MEP amplitudes following PASN20. Wu et al. (2012) also reported “substantial variability” in their GTS sample using iTBS to induce LTP-like plasticity, but did not report whether absolute changes in amplitude were similar in GTS patients and healthy controls Two studies, using iTBS and HFS, have previously shown reduced synaptic plasticity in GTS patients with comorbidities compared to healthy controls 25 but that it enhanced motor learning one week later 25-induced long-term structural changes in M1. The present study confirms this finding by showing that synaptic plasticity was unrelated to motor learning 45 min after PASN20 but correlated positively with motor learning in healthy controls 9 months after PASN20. The results indicate a long-term beneficial effect of induced LTP-like plasticity in healthy controls. However, GTS patients did not express LTP-like changes in response to PASN20.The main question addressed in the present study was whether synaptic plasticity can be related to impaired motor learning in GTS patients. Our data suggests that aberrant synaptic plasticity in GTS is related to reduced long-term consolidation of motor skills in the rotary pursuit task. Rajji and colleagues (2011) found that TMS induced LTP did not enhance performance in the rotary pursuit task 45 min after PASN20. In other words, long-term consolidation of motor learning appeared to be stronger in healthy controls than in GTS patients. These results could be accounted for in two different ways. Either long-term consolidation of motor learning in GTS patients is impaired in general, or GTS patients did not benefit from PASN20 because no LTP was induced. However, if long-term consolidation of motor learning is generally impaired in GTS patients, this may also be related to aberrant synaptic plasticity. TMS-induced LTP is thought to rely on the same biological processes as learning experiences in a natural environment N20, they may also differ with respect to biological processes in motor learning tasks. This would be an interesting question to address in an independent experiment.Although motor skill acquisition in the rotary pursuit task was normal in GTS patients Suppa and colleagues (2011) and Wu and colleagues (2012) have pointed out that synaptic plasticity in GTS patients may be altered because of meta-plasticity effects, which may occur as a consequence of tics N20 stimulation than do healthy brains.According to the Bienenstock-Cooper-Munro rule, there is a floating threshold, which determines the amount of activity needed to elicit LTP or LTD. The activation needed is a function of the average postsynaptic activity levels If there is a long-term compensatory mechanism, it might not be as simple as single cell threshold adaptation though. Most GTS patients gain increased control over their symptoms during adolescence Several findings in the present study would support the assumption of a compensatory mechanism. Remarkably, LTD-like changes were strongly associated with fewer premonitory urges and fewer tics. Fewer tics were in turn associated with fewer premonitory urges. Moreover, those GTS patients who reacted with LTD-like plasticity instead of LTP-like plasticity were better at motor skill acquisition in the rotary pursuit task. If LTD-like plasticity were indeed a compensatory mechanism, then these results would indicate that patients who compensate more successfully for their tics and urges may also develop strategies in dealing with motor learning more successfully.An alternative explanation for LTD-like plasticity in GTS would have been an increased cortical excitability at baseline reflecting a homeostatic reaction of an „overexcited“ brain. Reversed effects of TMS protocols that normally induce LTP, such as PAS, occur in healthy volunteers if cortico-spinal excitability is altered at baseline and have been attributed to homeostatic meta-plasticity Another finding in this study was that IO curves did not differ between the groups at baseline, which is an interesting result with respect to previous inconsistent IO findings in GTS. While one study found a shallower slope in GTS patients as compared to healthy participants, suggesting reduced cortico-spinal excitability at rest N20 responses but this is unlikely because no tics occurred during the assessments of MEPs and IO curves. We cannot exclude the possibility though that ticcing or tic suppression at other times during the experimental procedure influenced results. Furthermore, we cannot deduce whether the difference in long-term consolidation between the groups is a general problem in GTS or whether it was due to the absence of induced LTP at time 1. Further research will be needed to determine whether motor skill consolidation is generally impaired in GTS patients. Moreover, it would be interesting to induce synaptic plasticity in children with GTS in order to investigate whether they show a normal response to PAS. If our findings of aberrant plasticity in GTS reflect a compensatory mechanism, it should not be present before puberty.The main limitation of this study is its small sample size. The population investigated was very homogeneous though, making it more likely that the results can be generalized to other uncomplicated GTS patients despite the sample size. However, approximately 90% of GTS patients suffer from comorbidities N20 differed between a small sample of GTS patients and healthy controls. The majority of patients responded with LTD-like changes, while the majority of healthy participants responded with LTP-like changes. Patients also showed reduced motor skill consolidation as compared to healthy controls 9 months after PASN20. Overall, patients may not benefit from the long-term structural changes that occur when LTP-like effects are induced. Although LTP was artificially induced in this study these results may help to explain impairments in cortex-based motor learning in GTS patients more generally. Moreover, GTS patients with strong premonitory urges and more severe tics tended to show physiological LTP-like plasticity. In contrast, less severely affected patients had LTD-like responses, suggesting a compensatory mechanism.Synaptic plasticity in response to PASTable S1Correlations between physiological measures. Correlations between the input-output (IO) slopes before the paired associative stimulation , immediately after PAS (time 2) and 30 min. after PAS (time 3) with motor evoked potential (MEP) changes from time 1 to time 2 and the resting motor threshold in Gilles de la Tourette (GTS) patients and healthy controls. Significance levels: *p<.05.(DOCX)Click here for additional data file.


These findings may guide the development of a novel class of disease-modifying drugs with anti-inflammatory and anti-nociceptive effects.We demonstrate a novel dual strategy against inflammation and pain through body-wide desensitization of nociceptors via TRPA1. Attenuation of experimental colitis by capsazepine (CPZ) has long been attributed to its antagonistic action on TRPV1 and associated inhibition of neurogenic inflammation. In contrast, we found that CPZ exerts its anti-inflammatory effects via profound desensitization of TRPA1. Micromolar CPZ induced calcium influx in isolated dorsal root ganglion (DRG) neurons from wild-type (WT) but not TRPA1-deficient mice. CPZ-induced calcium transients in human TRPA1-expressing HEK293t cells were blocked by the selective TRPA1 antagonists HC 030031 and A967079 and involved three cysteine residues in the N-terminal domain. Intriguingly, both colonic enemas and drinking water with CPZ led to profound systemic hypoalgesia in WT and TRPV1 Sensory neurons of nociceptive character, which encode chemical, thermal, and mechanical stimuli and release neuropeptides, pervade the whole mammalian body. These nerve fibers are equipped with polymodal ion channel receptors, which are members of a large family, including the two major irritant receptor-channels transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1)123236789101213147In this study we show that TRPA1 agonism, rather than TRPV1 inhibition, in colonic sensory neurons is the key initial step in colitis protection by CPZ enemas. Intriguingly, colonic and systemic administration of CPZ or AITC induced a profound body-wide, TRPA1-mediated desensitization of nociception in mice. Chronic administration of these TRPA1 agonists was well tolerated. Thus, systemic desensitization through TRPA1 may provide a novel strategy for medicinal treatment of various chronic inflammatory and pain states.−/− mice, which was reflected by an improved endoscopic score and reduced loss of body weight  colitis in wild-type (WT) and TRPV1-deficient mice (each group n = 8). Although conflicting reports exist, TRPV1-deficient mice developed DSS colitis and weight loss to the same degree as the congenic WT mice in our laboratory, which complies with our results from the model of TNBS colitis that we had previously published7891011y weight . H&E stay weight . In accoy weight .in vivo observation that CPZ enemas effectively inhibited colitis in TRPV1 null mutant mice imposed the question on TRPV1-independent neuronal effects of CPZ. Since TRPA1 had been shown to be crucial in various models of inflammation including colitis612The Patch clamp experiments were carried out in voltage clamp mode on HEK293t cells expressing recombinant hTRPA1 (hTRPA1-HEK293t cells). At a holding potential of −60 mV, CPZ (10 μM) induced an inward current that was almost completely inhibited  by the selective TRPA1 antagonist HC-030031  . In all Selective action of CPZ on TRPA1 was confirmed by employing the calcium microfluorimetry technique. hTRPA1-HEK293 cells were stimulated by two applications of CPZ (50 μM) for 10 s at 5-min intervals . The sel50 value estimated to 30 μM CPZ and the small AITC response after 100 μM CPZ again suggested cross-desensitization , while none of the 448 TRPA1-deficient DRG neurons tested showed calcium influx in response to CPZ (100 μM) as illustrated by representative examples in DRG neurons were maintained in primary culture and investigated by calcium microfluorimetry. Cells were stimulated by CPZ , followed by AITC , capsaicin  and KCl . tization . Figure −/− mice, when these mice were challenged by CAP instillations into the eye  were obviously painful in the WT mice. We then quantified the CPZ (531 μM) enema-induced nocifensive behavior in healthy animals (each n = 6) by counting the writhing reactions and recording visceromotor reflex responses (VMRs) through the integrated electromyography (EMG) of the abdominal muscle wall . During  the eye .Since repeated enemas are an unpleasant route of administration, we tested for a possible anti-nociceptive effect of peroral CPZ (531 μM) in the drinking water. The drinking regimen over 10 days was well tolerated with no obvious adverse effects occurring. During the course of continuous oral CPZ administration paw-withdrawal latency to radiant heat stimulation (Hargreaves´ method) progressively increased in both hindpaws . The tolin vivo after the final enema. TRPV1 mRNA did not change under both circumstances. Thus, transcriptional upregulation of the TRPA1 gene expression had taken place, due to the multiple CPZ enemas, but was rapidly reversed when the CPZ supply was discontinued. Under DRG culturing conditions, the same epigenetic reversal had likely taken place and all residual CPZ was probably washed-out, so that no residual desensitization could actually be expected. As the cellular models did not reflect the outlasting CPZ-induced desensitization of the whole animals in vivo, we looked for another strong indicator of the surprising systemic effect. The majority of nociceptive neurons is peptidergic, expressing predominantly calcitonin gene-related peptide (CGRP) and substance P (SP)151920in vivo, twice daily for 7 d were isolated and tested 12–16 h after the last enema. Neither CPZ (100 μM) nor AITC (100 μM) induced any CGRP release in this condition  and TRPV1 (CAP) agonists were not significantly different in DRG neurons from control animals and enema-treated animals . Howeverondition ; notablyondition . In contIt has been assumed that inhibition of TRPV1 in peptidergic colonic sensory neurons by CPZ attenuates colitis through reducing neuropeptide release and neurogenic inflammation. In stark contrast, we found that the activation of TRPA1 by CPZ followed by profound and sustained desensitization caused the anti-inflammatory effect. More importantly, we discovered an unexpected powerful impact of CPZ via TRPA1 on peripheral nociception in general. To the best of our knowledge the present study is the first to demonstrate body-wide nociceptor desensitization through TRPA1.Previously, one dermatological study suggested that local TRPA1 desensitization by AITC suppressed the inflammatory reaction in an allergic contact sensitivity model of the mouse67Our patch clamp and calcium imaging measurements demonstrate a concentration-dependent activation of heterologously expressed human TRPA1 by CPZ. Activation of TRPA1 by electrophiles, such as AITC or TNBS, occurs through covalent modification of cysteine residues in the N-terminal domain of the channel4in vivo with repeated CPZ enemas and CPZ or AITC in the drinking water.The calcium microfluorimetry measurements in lumbosacral DRGs showed an almost complete overlap 93%) of the chemosensitivity to CPZ and to AITC at comparable concentrations. As in several recent publications the overlap between capsaicin- and AITC-sensitive DRG neurons was not as complete as erstwhile postulated252612% of the 282930In vivo, the lipophilic drug may be better retained in the tissues or re-distributed. Some support for this interpretation comes from additional experiments quantifying the mRNA for TRPA1 and TRPV1 in DRGs. The ganglia were either isolated and processed immediately after the final CPZ enema or 24 h later. TRPA1 gene expression was clearly enhanced in the former case but normal after one day of survival, indicating a rapid reversal of the epigenetic effect. Our behavioral experiments still indicate that CPZ induced a TRPA1-driven local desensitization of colonic sensory neurons, since TRPA1-deficient mice did not show the initial nocifensive responses during and after the CPZ enema administrations. By contrast, WT mice gradually lost their pain responses . Intriguingly, desensitization did not remain a local, colonic phenomenon but was extended – supposedly by absorption and systemic distribution of the drug – to such distant organs as the eye, where drops of CAP or AITC solution were no longer able to evoke the eye-wiping reaction, or as the skin, where these irritants induced only minimal neuropeptide release. The results from the radiant heat plantar test after oral administration of CPZ and AITC further support the notion of a body-wide, general desensitization of nociceptive nerve endings via TRPA1 without affecting mechanosensitivity to non-noxious stimuli. Since AITC application via drinking water had effects similar to those after oral administration of CPZ, we suggest a class effect of TRPA1 agonists. It remains possible that the stronger effect of CPZ compared with AITC was due to the simultaneous TRPV1 antagonism of CPZ.The sustained desensitization of stimulated CGRP release from isolated skin and colon after CPZ enemas cannot be attributed to a disruption of the vesicular exocytotic release mechanism, because this remained undisturbed as evidenced by KCL-induced unspecific depolarization. Thus, the excitability with respect to the function of voltage-gated calcium channels in the sensory nerves was well maintained. Excitability with respect to action potential triggering does not matter in our models, as TRP channel-induced CGRP release cannot be altered even by TTX and lidocain18As a secondary finding, the feasibility of the oral administration route of CPZ and AITC, its apparent tolerability, safety, and efficacy can be postulated. Higher concentrations of CPZ may even support the anti-nociceptive effect of the chronic desensitization. Even the original target effect of CPZ, blocking the CAP binding site in TRPV1, may be adjuvant because it would preclude effects of inflammatory endovanilloids637Our findings provide novel insight into the molecular mechanisms that lead to colitis attenuation by CPZ and strengthen the crucial role of TRPA1 as a gatekeeper for inflammation. Moreover, we demonstrate a novel mechanism to induce body-wide anti-nociception through TRPA1 desensitization. This dual mechanism may provide new therapeutic options not only for patients with inflammatory or irritable bowel disorders but also for patients suffering from other chronic states of pain or inflammation. TRPA1 desensitization could not only provide symptomatic relief but it could also support real cure, potentially enabling CPZ or derived novel “TRPA1 desensitizers” to become disease-modifying-drugs. Subjects with demonstrated genetic susceptibility to inflammatory or irritable bowel disorders could even benefit from a preventive approach.SI Materials and Methods. All animal experiments were performed in accordance with the relevant guidelines and regulations approved by the Animal Protection Authority, District Government Mittelfranken, Ansbach, Germany.For details see Whole-cell voltage clamp experiments were carried out in HEK293 cells transiently transfected with human TRPA1 (hTRPA1) using an Axon Axopatch 200B patch clamp amplifier and the pCLAMP 10 software .Mouse lumbosacral DRG neurons were isolated and cultured as previously described, using nerve growth factor Experimental colitis was induced by Dextran Sulphate Sodium  which was added to the drinking water for 7 days. Monitoring of the course of colitis was performed by daily body weight measurements and mouse colonoscopies on day 2, 4 and 6. To assess colitis severity, previously established endoscopical and histological score systems were used6Mice received capsazepine  or vehicle (PBS) enemas twice daily for seven days under brief isoflurane anesthesia. In another experiment the same concentration of CPZ (in 0.1% DMSO) or AITC (500 μM)  was continuously applied through the drinking water.All experiments were done in compliance with the local Animal Protection Authority . Animals were randomized and investigators were blind to the different groups of animals and the handling of data (treatment vs. control). The writhing test, eye-wipe, von-Frey and Hargreaves’ test as well as measurements of visceromotor responses were performed as published before404142How to cite this article: Kistner, K. et al. Systemic desensitization through TRPA1 channels by capsazepine and mustard oil - a novel strategy against inflammation and pain. Sci. Rep.6, 28621; doi: 10.1038/srep28621 (2016).


Avid renal sodium and water retention among other mechanisms produce ascites in patients with cirrhosis. The main guidelines recommend sodium intake reduction in order to counteract this complication. However, some randomized controlled trials have suggested a lack of benefit with a sodium-restricted over an unrestricted diet, and even an increase in ascites and renal complications has been reported. There are no systematic reviews addressing this question.A systematic review protocol has been designed and will be reported in line with Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P). We will search for randomized controlled trials evaluating a salt-restricted versus unrestricted regime in patients with cirrhosis and ascites in EMBASE, MEDLINE, and the Cochrane Central Register of Controlled Trials. We will also try to identify literature by reviewing reference list of included studies and relevant reviews, screening main conference proceedings, and searching for unpublished and ongoing trials in the World Health Organization (WHO) International Clinical Trials Registry Platform.Two researchers will independently undertake selection of studies, data extraction, and assessment of the quality of included studies.We will estimate pooled risk ratios for dichotomous data and the mean difference or standardized mean difference for continuous outcomes. A random effect model will be used for meta-analyses. Data synthesis and other analyses will be conducted using RevMan software. Ethics and dissemination: no ethics approval is considered necessary. Results of this study will be disseminated via peer-reviewed publications and social networksSodium restriction is a widely accepted coadjuvant therapy for ascites; however, this indication is based primarily on expert recommendations. As far as we know, this will be the first systematic review assessing the effects of a sodium-restricted diet for ascites in cirrhotic patients.Our systematic review will aim to provide a high-quality synthesis of current evidence for patients and clinicians about this question.The main limitation might result from the reduced number and quality of primary studies available.CRD42015022161PROSPERO The online version of this article (doi:10.1186/s13643-016-0250-4) contains supplementary material, which is available to authorized users. Hepatic cirrhosis represents the final stage of progressive and irreversible liver fibrosis observed in chronic hepatic disease. Liver architecture distortion determines portal hypertension. Added neurohumoral changes produce peripheral vasodilation, increased circulating volume, and sinusoidal vasoconstriction. The exact mechanism of peripheral vasodilatation is still unclear, but apparently, it is due to an imbalance between vasoconstrictors and vasodilators released as response to increased portal vascular resistance . CirculaAscites occurs as a consequence of these factors, generating a wide range of problems that affect life expectancy starting with general disability, dyspnea, peritoneal infections, renal failure, and finally death .Salt restriction appears as an obvious way to avoid sodium retention and water overload.It is usually posited that in order to avoid fluid retention, oral sodium intake must be significantly reduced in order to achieve a negative sodium balance. Salt restriction would further decrease fluid accumulation by lowering portal pressure through vascular volume depletion.All major guidelines in this topic recommend salt restriction in these patients.However, some authors warn that patients with cirrhotic ascites have low blood sodium levels; therefore, the use of diuretics and sodium restriction could decrease renal perfusion, resulting in further renal impairment and more ascites. Consequently, some randomized controlled studies have suggested there would be no benefit, and even harm, with a sodium-restricted over an unrestricted diet , 6.There are no completed or ongoing systematic reviews on this topic. Therefore, it is critical to provide a high-quality synthesis of current evidence for patients and clinicians about this question.The aim of this systematic review is to assess the effects of a salt-restricted diet for patients with cirrhotic ascites.A systematic review protocol has been designed and will be reported in line with Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P)  in EMBASE, MEDLINE, and the Cochrane Central Register of Controlled Trials.#1 randomized controlled trial [pt]#2 controlled clinical trial [pt]#3 randomized [tiab]#4 placebo [tiab]#5 drug therapy [sh]#6 randomly [tiab]#7 trial [tiab]#8 groups [tiab]#9 #1 OR #2 OR #3 OR #4 OR #5 OR #6 OR #7 OR #8#10 animals [mh] NOT humans [mh]#11 #9 NOT #10#12 "Sodium, Dietary"[Mesh]#13 "Diet, Sodium-Restricted"[MeSH]#14 "Sodium Chloride"[Mesh]#15 "Sodium"[Mesh]#16 salt[ti] OR sodium[ti]#17 (restrict* OR low OR reduc* OR intak* OR added OR diet OR consum* OR excess* OR increas* or high) AND # 18 #12 OR #13 OR #14 OR #15 OR #16 OR #17#19 "Liver Cirrhosis"[Mesh]#20 (hepatic or liver) and (fibrosis or cirrho* OR ascit*)#21 #19 OR #20#21 #11 AND #18 AND #21The following strategy will be used to search MEDLINE (PubMed) and adapted for other sources:Review clinical practice guidelines and relevant reviews for potentially eligible studiesScreen the list of references of relevant reviews and included studies identifiedManually review proceedings of major conferences in the areaSearch for ongoing studies in the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP)Additionally, we will do the following:www.covidence.org).Deduplicated records will be uploaded to Covidence Software  general information of the study; (2) study characteristics ; (3) participants ; (4) interventions and controls ; (5) outcomes ; and (6) results.Two review authors will independently extract data in pre-designed data extraction and validity assessment forms. A third review author will act as an arbiter in case of disagreement. The methodological quality will be assessed with the Cochrane Collaboration’s tool for assessing risk of bias (Cochrane Handbook (V.5.1.0)), by the following criteria: (1) randomization; (2) allocation concealment; (3) blinding; (4) data integrity; (5) selective reporting; and (6) other bias, such as trial design, baseline similarity of groups, and early cessation of treatment. For all of the studies, the assessment should follow the above six criteria and be categorized as low, unclear, or high risk of bias.Dichotomous data will be determined by using a risk ratio (RR) with 95 % confidence interval (CI), whereas continuous outcomes will be analyzed using mean differences (with 95 % CI) or standardized mean differences (95 % CI) if different measurement scales are used.Q statistic) and the I2 statistic. Statistically significant heterogeneity will be defined as at least one positive test (either P < 0.10 using the Mantel-Haenszel chi2 test or >50 % using the I2 statistic).We will assess heterogeneity quantitatively with a formal statistical test (If possible, all trials will be combined in a meta-analysis comparing salt-restricted diet versus unrestricted diets. A random effects model will be used for all the analyses. Separate meta-analyses will be presented for specific populations or interventions if statistically significant heterogeneity is explained by some of these or if a convincing subgroup effect is found.We will perform statistical analysis in accordance with the guidelines for statistical analysis developed by The Cochrane Collaboration, using the statistical software RevMan 5.3.Ascites severityCirrhosis severityThe following subgroups will be investigated, if possible:We will perform sensitivity analyses to address the impact of studies with higher or lower bias risk.P < 0.10 and present intercepts with 90 % CIs. Outcome reporting bias will be evaluated through discrepancies between the registered protocol and the final publication. We will contact authors for more information if we are not able to find the record of a study in the trials registries.We will investigate the publication bias visually with the use of funnel plots. We will base evidence of asymmetry on We will judge the certainty of the evidence for all outcomes using the Grading of Recommendations Assessment, Development, and Evaluation working group methodology .This systematic review will synthesize scientific evidence for the effect of diet restriction sodium on ascites.As far as we know, this will be the first systematic review to assess the effectiveness and safety of sodium-restricted diet for ascites in cirrhotic patients. It aims to provide a high-quality synthesis of the current evidence for patients and clinicians about this question.The main limitation might result from the reduced number and quality of primary studies available. Another limitation might arise from the lack of standardization in the definition of ascites and treatment success or failure. The findings from this systematic review will be valuable in the decision-making process about the effect of sodium-restricted diet as therapy for ascites.


Electrical stimulation is a common adjunct used to promote bone healing; its efficacy, however, remains uncertain. We conducted a meta-analysis of randomized sham-controlled trials to establish the efficacy of electrical stimulation for bone healing. We identified all trials randomizing patients to electrical or sham stimulation for bone healing. Outcomes were pain relief, functional improvement, and radiographic nonunion. Two reviewers assessed eligibility and risk of bias, performed data extraction, and rated the quality of the evidence. Fifteen trials met our inclusion criteria. Moderate quality evidence from 4 trials found that stimulation produced a significant improvement in pain (mean difference (MD) on 100-millimeter visual analogue scale = −7.7 mm; 95% CI −13.92 to −1.43; p = 0.02). Two trials found no difference in functional outcome . Moderate quality evidence from 15 trials found that stimulation reduced radiographic nonunion rates by 35% . Patients treated with electrical stimulation as an adjunct for bone healing have less pain and are at reduced risk for radiographic nonunion; functional outcome data are limited and requires increased focus in future trials. Bone healing is a complex physiological process and is the end goal in the treatment of patients with fractures, surgical osteotomies and spinal fusion procedures. Failure or delays in bone healing often require further intervention and may result in serious morbidity such as increased pain and functional limitations23Electrical stimulation is a popular adjunctive therapy used to promote bone healing across a range of indications537891012131415161718Cochrane Handbook for Systematic Reviews of InterventionsWe report this study according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statementWe systematically searched MEDLINE, EMBASE, CINAHL, and the Cochrane Library from inception of the database to March 6, 2016. We used MeSH and EMTREE headings in various combinations and supplemented with free text to increase sensitivity . Manual Two authors independently screened all titles and abstracts and applied eligibility criteria to the methods section of potentially eligible trials using an electronic screening form. All discrepancies were resolved by consensus.We included all studies fulfilling the following criteria:1) Adult patients >16 years of any sex undergoing operative or nonoperative treatment for a fresh fracture, nonunion, delayed union, osteotomy, or symptomatic spinal instability requiring fusion.2) Trials comparing direct current (DC), capacitive coupling (CC), or pulsed electromagnetic field therapy (PEMF).3) Randomized sham-controlled trials (RCT) onlyNo restrictions were made for publication date, language, presence or absence of co-interventions, or length of follow-up. Studies using multiple bones in the same patients as the unit of randomization, rather than patients were excluded due to lack of independenceTwo reviewers independently performed outcome-specific assessment of risk of bias using the Cochrane Collaboration’s tool for risk-of-bias assessmentTwo reviewers independently extracted data using a piloted electronic data extraction form. Extracted data included author names, journal names and publication year, funding source, sample size, mean ages and proportion of each sex in treatment and control groups, descriptions of the interventions in each group, all reported outcomes and follow-up times, and loss to follow-up. We attempted to contact study authors for clarification if important data were unclear or not reported.Radiographic healing was determined according to the methods implemented in each trial. When multiple criteria for union were described, we recorded the most conservative estimate of union. For each trial we determined whether radiographic assessment was blinded or independently assessed and judged by consensus whether the determination of union was reasonable or not reasonable. We converted radiographic union rates to the number of nonunions by subtracting from the total number of patients in each group. For patients already presenting with a nonunion or delayed union, we recorded the number of patients with persistent or on-going nonunions. In trials that reported union based on CT-scan and plain radiographs, we recorded plain film radiographic healing for consistency.We calculated agreement for reviewers’ assessments of study eligibility with the Cohen’s kappa coefficient and agreement for assessments of risk of bias using the intraclass correlation coefficient (ICC). Kappa values ≥0.65 were considered adequateAmong eligible trials we found substantial diversity in the types of bone lesions targeted for treatment. Although baseline bone healing time differs by size of bone and the site of lesion, the biologic process of healing is consistent across all bone lesions262728p-values of <0.05 were considered significant.We utilized the conservative random-effects model of DerSimonian and Laird to pool effect estimates213221Χ2test for heterogeneity and the I2 statisticI2 values were interpreted according to the Cochrane HandbookClinical indication: fresh fractures, delayed union or nonunion, spinal fusion, or surgical osteotomy.Type of stimulation: direct current (DC), capacitive coupling (CC), or pulsed electromagnetic fields (PEMF).We quantified heterogeneity using the For each subgroup, we performed tests for interaction using a chi-square significance testOur main reported analysis is a complete case analysis in which participants with missing data were excluded from both the numerator and denominator. To explore the effects of missing outcome data, we performed sensitivity analyses. For the control group, we assumed the event rate to be the same for patients with missing data and those successfully followed; for the treatment group we assumed plausible ratios of event rates in patients with missing data compared with those who were successfully followed at ratios of: 1.5:1, 2:1, and 2.5:137Given potential variability in the methods used to evaluate radiographic union3940We utilized the GRADE approach to summarize the quality of the evidence for or against the use of electrical stimulation by each outcome. Data from randomized controlled trials were considered high-quality evidence, but could have been rated down according to risk of bias, imprecision, inconsistency, indirectness, or publication biaset al. reported different outcomes of the same patient population in two separate publications45444546474849505152535455565758Of 2025 potentially eligible articles, 1664 titles and abstracts were screened and 17 were eligible for our review. However, the authors of one trialMean age of study participants was 45 years in the experimental and control arm. The proportion of male patients in the experimental and control arm was 58.3% and 56.3%, respectively. Mean follow-up was 8.2 (SD 3.4) months for radiographic outcomes and 8.6 (SD 3.7) months for pain and function .Four trials included patients undergoing a spinal fusion4546495244475051544856575847485354575056Twelve trials reported use of pulsed electromagnetic field (PEMF) therapy, one trial reported direct current (DC) stimulation and two trials reported continuous current (CC) stimulation. Details with regards to specific stimulator type, frequency and treatment duration for each study are reported in Radiographic union was described in all 15 of the included trials . Consens485456Pain was reported in four trials using either the Visual Analogue Scale (VAS)4846Risk of bias assessments are presented in 2 = 0%; Pain was reported across four trials4446482 = 57%)Functional outcomes were compared in two trials that reported SF-36 scores (n = 316 patients). The pooled estimate of effect between electrical stimulation and control was not statistically significant  . In the 462 = 46%; p = 0.02) ((Appendices 5 and 6). An analysis conducted with the spine studies removed still showed a significant pooled treatment effect in favor of electrical stimulation for acute fracture, nonunion or delayed union, and osteotomy .Radiographic nonunion was compared across 15 trials with 1247 patients. The pooled estimate of effect between electrical stimulation and sham controls at last reported follow-up up to 12 months found that electrical stimulation reduced the relative risk for nonunion or persistent nonunion by 35%  and the absolute risk by 15%. Overall between-study heterogeneity was moderate (I = 0.02) . Interpr = 0.02) . We founOur complete case analysis showed a significant difference  that remained robust when assumed nonunion rates in patients with missing data were 1.5:1 and 2:1. When the assumed nonunion rates in patients with missing data went up to 2.5:1 the pooled estimate of effect was no longer significant .In only trials in which an independent assessor was used to determine union, a significant difference in favor of electrical stimulation was found . Trials in which consensus judgment deemed the definition and assessment as reasonable showed a significant difference in favor of electrical stimulation . Finally, the most conservative estimate in only those trials44475051525357Our systematic review and meta-analysis of eligible randomized controlled trials found moderate quality evidence for electrical stimulation in reducing patient-reported pain and radiographic nonunion or persistent nonunion. Low-quality functional outcome data showed no difference with electrical stimulation compared to sham treatment.Our findings are strengthened by our comprehensive search and broad clinical eligibility criteria, and by including only randomized sham-controlled trials. We hypothesized the effect of electrical stimulation on bone healing would be similar across different types of stimulation and different clinical lesions, and our subgroup analyses support this assumption. We found no evidence to support a difference in treatment effect due to treatment indication (interaction p = 0.75). In keeping with other orthopaedic trials of bone healing, most trials reported only surrogate end points. Limited reporting of patient-important outcomes is highlighted in this review and has been identified as a significant problem in the surgical literature6365et al.51et al. conducted a meta-analysis looking at the efficacy of various types of electrical stimulation on spinal fusionA Cochrane review published in 2011 reported non-significant differences for electrical stimulation in improving union rates in four trials involving 125 patientsA previous review performed by our group in 2008434447505154A survey of 450 Canadian trauma surgeons in 2008 (response rate 79%) demonstrated that 23% of surgeons used electrical bone stimulators to accelerate bone healingThis systematic review and meta-analysis found that patients treated with electrical stimulation as an adjunct for bone healing have significantly less pain and experience lower rates of radiographic nonunion or persistent nonunion. No difference was seen with regards to functional outcomes in a limited number of trials. Future trials focusing on functional outcomes to identify appropriate indications and ideal patient selection are warranted.How to cite this article: Aleem, I. S. et al. Efficacy of Electrical Stimulators for Bone Healing: A Meta-Analysis of Randomized Sham-Controlled Trials. Sci. Rep.6, 31724; doi: 10.1038/srep31724 (2016).


The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA . Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues  RNA interference (RNAi) is a highly conserved endogenous cellular process whereby small interfering RNAs (siRNA) regulate post transcriptional gene expression via sequence-specific mRNA degradation and translational blocking. Due to its potent specific gene knockdown effects and efficiency, RNAi has emerged as a promising approach to treat molecular targets in a spectrum of human disease, including cardiac disease , but cliin vitro and in vivo [Custom designed microbubbles (MBs) are emerging as gene therapy vectors that may overcome several limitations of current RNAi delivery platforms and also function as true theranostic agents –6. MBs c in vivo , 14–17.We tested the hypothesis that a non-invasive and non-viral nucleic acid delivery platform utilizing MBs and ultrasound could mediate RNAi in the heart. To prove this concept, we used a customized MB formulation and acoustic regimen for cardiac-specific luciferase siRNA delivery in a transgenic mouse with cardiac-restricted expression of luciferase. Cardiac expression of luciferase was assayed at the RNA and protein levels.Transgenic mice with cardiac-restricted expression of luciferase were produced by a commercial vendor through microinjection of the luciferase transgene into fertilized mouse egg nuclei  which was maintained in FVB (Charles River Laboratories) mice. 8 MB/mL with a mean diameter of 2.1±1.1 μm as measured by a Coulter Multisizer 3 (Beckman Coulter).Perfluorobutane gas-filled lipid MBs carrying either Silencer Select siRNA (Ambion) against luciferase or a negative control sequence were prepared as previously described , 17. BriAll animal procedures conformed to NIH guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. For all surgical procedures, mice were anesthetized using 1–2% inhaled isoflurane and the depth of anesthesia was monitored by toe pinch response. All mice were euthanized by resection of the heart and decapitation under deep isoflurane anesthesia (5%).While mice were under anesthesia a venous cannula was surgically placed in the jugular vein for MB infusion. To shield the lungs from ultrasound, acoustic absorbing material with a circular window (10 mm diameter) overlying the heart was placed on the chest , with caA 0.5–0.7 mL suspension of MBs loaded with either luciferase siRNA (n = 9) or negative control siRNA (n = 8) was infused over 15 minutes during simultaneous treatment with the ultrasound regime described above. Ultrasound treatment was continued for an additional 5 minutes after the end of the infusion . After treatment, the venous cannula was removed and the mice were recovered. Three days thereafter, the mice were euthanized as described above, and tissues were excised for measurement of luciferase mRNA and protein levels.To determine if UTMD causes tissue injury, hearts and lungs were harvested from 3 additional mice 1.5 hours after UTMD treatment and fixed in formalin. As a negative control, the heart and lungs were also harvested from a single mouse that did not receive UTMD treatment. Tissue sections were stained with hematoxylin and eosin (H&E) and read by a veterinary pathologist blinded to experimental condition to identify any evidence of hemorrhage, petechiae, or other treatment-related injury such as necrosis.Ventricles were harvested post-mortem to quantify luciferase expression in 14 untreated mice in order to establish baseline levels of luciferase expression in target tissue. Tissue was collected from the atria, kidney, liver, lung, spleen and uterus in 5 of these 14 untreated mice to confirm cardiac restricted luciferase expression. All tissue samples were homogenized and protein content was estimated by Bradford assay. Luciferase activity was detected using a commercial assay (Promega) with a 20 second sampling time. Luciferase activity in treated mice was normalized to total protein and expressed as a percent of the mean luciferase activity detected in the ventricles of untreated mice. Data are reported as mean ± standard error.Ventricles from 8 of 14 untreated mice were processed for real time PCR to establish baseline levels of luciferase mRNA expression. A subset of the ventricles from luciferase siRNA treated mice (n = 8), and control siRNA treated mice (n = 7) were processed for real time PCR. RNA was extracted from tissue samples using Trizol reagent (Invitrogen), and complementary DNA was prepared from 2 μg of total RNA using Taqman reverse transcription kit (Applied Biosystems). Luciferase expression was determined by using RT PCR primers . The GAPDH gene  was used as a reference to normalize luciferase measurements. RT-PCR amplifications were performed with the Absolute blue Sybr Green/Rox kit (Thermo Scientific) using an Applied Biosystems 7900HT instrument.To further characterize the ultrasound cavitation phenomena associated with the treatment protocol, acoustic emissions from MB cavitation were recorded passively in a single separate mouse using a 3.5-MHz single-element transducer  with a 25 mm focal depth, positioned 25 mm from the heart and aligned to the focus of the treatment transducer both prior to, and during, MB infusion. The acoustic shield was removed to facilitate the alignment of the transducer and passive cavitation detector over the mouse heart. Acoustic emission signals were obtained upon ultrasound delivery as described above  before and during MB infusion. All received acoustic signals were filtered using a 2-MHz high pass filter to reduce the 1.3-MHz fundamental frequency, and amplified by 20 dB using a pulser/receiver in receive mode . Signals were digitized at a 25-MHz sampling frequency using a digital oscilloscope  and processed in Matlab (Mathworks). An 8-microsecond time gate was applied to each signal and a Fourier transform was performed to the time-domain signals in order to obtain the frequency domain spectra.−ΔΔCt method using untreated mice as the calibrator, and expressed as mean ± standard error. Luciferase expression at the mRNA and protein levels was not normally distributed and therefore a Mann-Whitney test was used to perform statistical comparisons between groups.Luciferase activity in luciferase siRNA-MB or control siRNA-MB treated mouse ventricles was normalized to the average luciferase activity found at baseline in the ventricles of non-treated mice and reported as mean ± standard error. Analysis of luciferase expression by RT-PCR was performed using the 2Luciferase activity was measured in different organs to confirm whether expression was restricted to the heart. High levels of luciferase activity were detected in cardiac tissue, with minimal activity detected in the kidney, liver, lung, spleen, or uterus .Ultrasound targeted microbubble destruction was observed in mouse hearts following infusion of luciferase-loaded MBs, as shown in p = 0.03, p<0.01, Three days after treatment, normalized luciferase protein activity was significantly reduced (by over 60%) in the ventricles of mice treated with luciferase siRNA-loaded MBs compared to ventricles of mice treated with control siRNA-loaded MBs  in 2 of 3 mice following UTMD treatment, falling within an area outside of our acoustic shield. There was no clinical evidence of respiratory distress following UTMD treatment. Representative images are shown in The excised heart and lung blocs appeared grossly normal, with no visual evidence of hemorrhage. H&E stained myocardial tissue sections were entirely normal, and there was no histologic evidence of myocardial injury or inflammation. There was a single focus of microscopic lung hemorrhage  and no clinical signs of respiratory distress were observed. Because the ultrasound beam used in this study encompassed the entire chest of the mice, a protective acoustic absorbing shield was fashioned, but will be less important in clinical applications, where the beam elevation is small relative to the size of the heart and ultrasound exposure of the lungs can be more easily avoided.Ultrasound at higher intensities is scattered by air in the lungs and this could produce pulmonary vascular hemorrhage, which could potentially be monitored with computed tomography . No grosin vitro [Several limitations to this study bear mention. We cannot definitively conclude that the luciferase silencing phenomenon observed in this study derived solely from the cardiomyocytes, as other cell types within the heart could have also expressed luciferase, such that the luciferase silencing that was measured could have been partially derived from these cell types. However, the phospholamban expression cassette used in this animal model has been shown to drive luciferase expression to high levels in cardiomyocytes in vitro , and we in vivo studies found that siRNA reduced luciferase expression in mice for up to 10 days in tumor cells and up to 3–4 weeks in non-dividing hepatocytes [The duration of luciferase knockdown following siRNA delivery was not assessed in this study. Previous atocytes , 31. ThiUltimately, UTMD could have therapeutic value in cardiac pathophysiologic states for which non-druggable gene targets have been identified. For example, other preclinical studies have previously demonstrated siRNA-mediated protection of the heart following myocardial infarction or ischemia/reperfusion injury by targeting genes such as Nox2 and HIF-1alpha-prolyl-4 hydroxylase-2, although non-invasive targeted delivery approaches were not employed , 33. ImpIn conclusion, MBs and ultrasound can deliver siRNA to the heart, resulting in gene expression knockdown. The use of a direct intravenous injection and application of external ultrasound is minimally invasive, inherently image-guided by virtue of ultrasound-enabled imaging and treatment, and amenable to serial treatments , 17. ThiS1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(ZIP)Click here for additional data file.