Identification of brain functional connectivity during acute transcutaneous tibial nerve stimulation: A 3T fMRI study.

OBJECTIVES: A feasibility proof-of-concept study was conducted to assess the effects of acute tibial nerve stimulation (TNS) on the central nervous system in healthy volunteers using functional magnetic resonance imaging (fMRI). MATERIALS AND METHODS: Fourteen healthy volunteers were included in a prospective, single-site study conducted on a clinical 3T MRI scanner. Four scans of functional MRI, each lasting 6 min, were acquired: two resting-state fMRI scans (prior and following the TNS intervention) and in-between two fMRI scans, both consisting of alternating rest periods and noninvasive acute transcutaneous TNS (TTNS). Whole brain seed-based functional connectivity (FC) correlation analysis was performed comparing TTNS stimulation with rest periods. Cluster-level familywise error (FWE) corrected p and a minimal cluster size of 200 voxels were used to explore FC patterns. RESULTS: Increased FC is reported between inferior frontal gyrus, posterior cingulate gyrus, and middle temporal gyrus with the precuneus as central receiving node. In addition, decreased FC in the cerebellum, hippocampus, and parahippocampal areas was observed. CONCLUSIONS: Altered FC is reported in areas which have been described to be also involved in lower urinary tract control. Although conducted with healthy controls, the assumption that the underlying therapeutic effect of TNS involves the central nervous system is supported and has to be further examined in patients with incomplete spinal cord injury.

Satisfaction with Homeopathic Service and Care for Persons with Spinal Cord Injury.

BACKGROUND: The aim of the study was to investigate the satisfaction of individuals with spinal cord injury (SCI) with a homeopathic service at an SCI rehabilitation center. PATIENTS AND METHODS: A cross-sectional questionnaire study was performed at an SCI rehabilitation center in Switzerland. It included patients with chronic SCI who presented themselves to a homeopathic service offered by the hospital in a 12-months period. The participants filled in standardized questionnaires in German: "Measure Yourself Medical Outcome Profile" (MYMOP), Treatment Satisfaction Questionnaire for Medication (TSQM-9), the European Project on Patient Evaluation of General Practice Care (EUROPEP) questionnaire, and a self-administered questionnaire. RESULTS: The data of 14 patients were analyzed. Symptom severity as well as bother by the symptoms that led to homeopathic treatment decreased under homeopathic treatment (severity: from 4.3 to 3.3; bother: from 4.2 to 2.9) and remained lower over time (severity: 2.6; bother: 2.7), suggesting a sustained effect. Irrespective of the test instrument used, satisfaction rates were higher for homeopathic service than for homeopathic medication, which was rated as successful by 50% of the participants. CONCLUSION: Persons with SCI suffering from secondary complications of SCI who accessed homeopathic care reported high satisfaction rates with the service. Therefore, homeopathic service can be considered as an additive measure in persons with SCI suffering from recurrent symptoms.Evaluierung der Zufriedenheit von Personen mit Querschnittlähmung (QSL) mit einer homöopathischen Betreuung an einem Rehabilitationszentrum für QSL.An einem Rehabilitationszentrum für QSL in der Schweiz wurde eine Querschnittserhebung mittels Fragebögen durchgeführt. Eingeschlossen wurden Personen mit chronischer QSL, die sich in einer von der Klinik angebotenen homöopathischen Sprechstunde in einem 12-Monats-Intervall vorstellten. Die Teilnehmenden füllten standardisierte Fragebogen in deutscher Sprache aus: "Measure Yourself Medical Outcome Profile" (MYMOP), Treatment Satisfaction Questionnaire for Medication (TSQM-9), den "European Project on Patient Evaluation of General Practice Care (EUROPEP)" Fragebogen sowie einen selbst-erstellten Fragebogen.Die Daten von 14 Teilnehmenden wurden ausgewertet. Der Schweregrad der Symptome sowie die Belastung durch die Symptome die zur homöopathischen Behandlung geführt haben, wurden unter der homöopathischen Therapie geringer (Schweregrad: von 4.3 auf 3.3; Belastung: von 4.2 auf 2.9) und blieben über den Untersuchungszeitraum geringer (Schweregrad: 2.6; Belastung 2.7), was einen anhaltenden Effekt nahelegt. Unabhängig von dem verwendeten Testinstrument waren die Zufriedenheitsraten für die homöopathische Betreuung höher als diejenigen für die homöopathische Medikation, die von 50% der Teilnehmenden als erfolgreich bewertet wurde.Personen mit QSL, die wegen Sekundärkomplikationen eine homöopathische Sprechstunde aufsuchten, berichteten eine hohe Zufriedenheit mit dieser Betreuung. Daher kann eine homöopathische Betreuung als zusätzliche Massnahme bei Personen mit QSL mit persistierender Symptomatik in Betracht gezogen werden.

Prominent microglial inclusions in transgenic mouse models of α-synucleinopathy that are distinct from neuronal lesions.

Alpha-synucleinopathies are a group of progressive neurodegenerative disorders, characterized by intracellular deposits of aggregated α-synuclein (αS). The clinical heterogeneity of these diseases is thought to be attributed to conformers (or strains) of αS but the contribution of inclusions in various cell types is unclear. The aim of the present work was to study αS conformers among different transgenic (TG) mouse models of α-synucleinopathies. To this end, four different TG mouse models were studied (Prnp-h[A53T]αS; Thy1-h[A53T]αS; Thy1-h[A30P]αS; Thy1-mαS) that overexpress human or murine αS and differed in their age-of-symptom onset and subsequent disease progression. Postmortem analysis of end-stage brains revealed robust neuronal αS pathology as evidenced by accumulation of αS serine 129 (p-αS) phosphorylation in the brainstem of all four TG mouse lines. Overall appearance of the pathology was similar and only modest differences were observed among additionally affected brain regions. To study αS conformers in these mice, we used pentameric formyl thiophene acetic acid (pFTAA), a fluorescent dye with amyloid conformation-dependent spectral properties. Unexpectedly, besides the neuronal αS pathology, we also found abundant pFTAA-positive inclusions in microglia of all four TG mouse lines. These microglial inclusions were also positive for Thioflavin S and showed immunoreactivity with antibodies recognizing the N-terminus of αS, but were largely p-αS-negative. In all four lines, spectral pFTAA analysis revealed conformational differences between microglia and neuronal inclusions but not among the different mouse models. Concomitant with neuronal lesions, microglial inclusions were already present at presymptomatic stages and could also be induced by seeded αS aggregation. Although nature and significance of microglial inclusions for human α-synucleinopathies remain to be clarified, the previously overlooked abundance of microglial inclusions in TG mouse models of α-synucleinopathy bears importance for mechanistic and preclinical-translational studies.

Amyloid polymorphisms constitute distinct clouds of conformational variants in different etiological subtypes of Alzheimer's disease.

The molecular architecture of amyloids formed in vivo can be interrogated using luminescent conjugated oligothiophenes (LCOs), a unique class of amyloid dyes. When bound to amyloid, LCOs yield fluorescence emission spectra that reflect the 3D structure of the protein aggregates. Given that synthetic amyloid-ß peptide (Aß) has been shown to adopt distinct structural conformations with different biological activities, we asked whether Aß can assume structurally and functionally distinct conformations within the brain. To this end, we analyzed the LCO-stained cores of ß-amyloid plaques in postmortem tissue sections from frontal, temporal, and occipital neocortices in 40 cases of familial Alzheimer's disease (AD) or sporadic (idiopathic) AD (sAD). The spectral attributes of LCO-bound plaques varied markedly in the brain, but the mean spectral properties of the amyloid cores were generally similar in all three cortical regions of individual patients. Remarkably, the LCO amyloid spectra differed significantly among some of the familial and sAD subtypes, and between typical patients with sAD and those with posterior cortical atrophy AD. Neither the amount of Aß nor its protease resistance correlated with LCO spectral properties. LCO spectral amyloid phenotypes could be partially conveyed to Aß plaques induced by experimental transmission in a mouse model. These findings indicate that polymorphic Aß-amyloid deposits within the brain cluster as clouds of conformational variants in different AD cases. Heterogeneity in the molecular architecture of pathogenic Aß among individuals and in etiologically distinct subtypes of AD justifies further studies to assess putative links between Aß conformation and clinical phenotype.

Aß seeding potency peaks in the early stages of cerebral ß-amyloidosis.

Little is known about the extent to which pathogenic factors drive the development of Alzheimer's disease (AD) at different stages of the long preclinical and clinical phases. Given that the aggregation of the ß-amyloid peptide (Aß) is an important factor in AD pathogenesis, we asked whether Aß seeds from brain extracts of mice at different stages of amyloid deposition differ in their biological activity. Specifically, we assessed the effect of age on Aß seeding activity in two mouse models of cerebral Aß amyloidosis (APPPS1 and APP23) with different ages of onset and rates of progression of Aß deposition. Brain extracts from these mice were serially diluted and inoculated into host mice. Strikingly, the seeding activity (seeding dose SD50) in extracts from donor mice of both models reached a plateau relatively early in the amyloidogenic process. When normalized to total brain Aß, the resulting specific seeding activity sharply peaked at the initial phase of Aß deposition, which in turn is characterized by a temporary several-fold increase in the Aß42/Aß40 ratio. At all stages, the specific seeding activity of the APPPS1 extract was higher compared to that of APP23 brain extract, consistent with a more important contribution of Aß42 than Aß40 to seed activity. Our findings indicate that the Aß seeding potency is greatest early in the pathogenic cascade and diminishes as Aß increasingly accumulates in brain. The present results provide experimental support for directing anti-Aß therapeutics to the earliest stage of the pathogenic cascade, preferably before the onset of amyloid deposition.

Conversion of Synthetic Aß to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.

UNLABELLED: The aggregation of amyloid-ß peptide (Aß) in brain is an early event and hallmark of Alzheimer's disease (AD). We combined the advantages of in vitro and in vivo approaches to study cerebral ß-amyloidosis by establishing a long-term hippocampal slice culture (HSC) model. While no Aß deposition was noted in untreated HSCs of postnatal Aß precursor protein transgenic (APP tg) mice, Aß deposition emerged in HSCs when cultures were treated once with brain extract from aged APP tg mice and the culture medium was continuously supplemented with synthetic Aß. Seeded Aß deposition was also observed under the same conditions in HSCs derived from wild-type or App-null mice but in no comparable way when HSCs were fixed before cultivation. Both the nature of the brain extract and the synthetic Aß species determined the conformational characteristics of HSC Aß deposition. HSC Aß deposits induced a microglia response, spine loss, and neuritic dystrophy but no obvious neuron loss. Remarkably, in contrast to in vitro aggregated synthetic Aß, homogenates of Aß deposits containing HSCs induced cerebral ß-amyloidosis upon intracerebral inoculation into young APP tg mice. Our results demonstrate that a living cellular environment promotes the seeded conversion of synthetic Aß into a potent in vivo seeding-active form. SIGNIFICANCE STATEMENT: In this study, we report the seeded induction of Aß aggregation and deposition in long-term hippocampal slice cultures. Remarkably, we find that the biological activities of the largely synthetic Aß aggregates in the culture are very similar to those observed in vivo This observation is the first to show that potent in vivo seeding-active Aß aggregates can be obtained by seeded conversion of synthetic Aß in a living (wild-type) cellular environment.

Distinct Spacing Between Anionic Groups: An Essential Chemical Determinant for Achieving Thiophene-Based Ligands to Distinguish ß-Amyloid or Tau Polymorphic Aggregates.

The accumulation of protein aggregates is associated with many devastating neurodegenerative diseases and the existence of distinct aggregated morphotypes has been suggested to explain the heterogeneous phenotype reported for these diseases. Thus, the development of molecular probes able to distinguish such morphotypes is essential. We report an anionic tetrameric oligothiophene compound that can be utilized for spectral assignment of different morphotypes of ß-amyloid or tau aggregates present in transgenic mice at distinct ages. The ability of the ligand to spectrally distinguish between the aggregated morphotypes was reduced when the spacing between the anionic substituents along the conjugated thiophene backbone was altered, which verified that specific molecular interactions between the ligand and the protein aggregate are necessary to detect aggregate polymorphism. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between different morphotypes of protein aggregates.

Endogenous murine Aß increases amyloid deposition in APP23 but not in APPPS1 transgenic mice.

Endogenous murine amyloid-ß peptide (Aß) is expressed in most Aß precursor protein (APP) transgenic mouse models of Alzheimer's disease but its contribution to ß-amyloidosis remains unclear. We demonstrate ∼ 35% increased cerebral Aß load in APP23 transgenic mice compared with age-matched APP23 mice on an App-null background. No such difference was found for the much faster Aß-depositing APPPS1 transgenic mouse model between animals with or without the murine App gene. Nevertheless, both APP23 and APPPS1 mice codeposited murine Aß, and immunoelectron microscopy revealed a tight association of murine Aß with human Aß fibrils. Deposition of murine Aß was considerably less efficient compared with the deposition of human Aß indicating a lower amyloidogenic potential of murine Aß in vivo. The amyloid dyes Pittsburgh Compound-B and pentamer formyl thiophene acetic acid did not differentiate between amyloid deposits consisting of human Aß and deposits of mixed human-murine Aß. Our data demonstrate a differential effect of murine Aß on human Aß deposition in different APP transgenic mice. The mechanistically complex interaction of human and mouse Aß may affect pathogenesis of the models and should be considered when models are used for translational preclinical studies.

Aß seeds resist inactivation by formaldehyde.

Cerebral ß-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts containing aggregated ß-amyloid (Aß) into young, pre-depositing Aß precursor protein- (APP) transgenic mice. Previous work has shown that the induction involves a prion-like seeding mechanism in which the seeding agent is aggregated Aß itself. Here we report that the ß-amyloid-inducing activity of Alzheimer's disease (AD) brain tissue or aged APP-transgenic mouse brain tissue is preserved, albeit with reduced efficacy, after formaldehyde fixation. Moreover, spectral analysis with amyloid conformation-sensitive luminescent conjugated oligothiophene dyes reveals that the strain-like properties of aggregated Aß are maintained in fixed tissues. The resistance of Aß seeds to inactivation and structural modification by formaldehyde underscores their remarkable durability, which in turn may contribute to their persistence and spread within the body. The present findings can be exploited to establish the relationship between the molecular structure of Aß aggregates and the variable clinical features and disease progression of AD even in archived, formalin-fixed autopsy material.

Spectral discrimination of cerebral amyloid lesions after peripheral application of luminescent conjugated oligothiophenes.

In vivo imaging of pathological protein aggregates provides essential knowledge of the kinetics and implications of these lesions in the progression of proteopathies, such as Alzheimer disease. Luminescent conjugated oligothiophenes are amyloid-specific ligands that bind and spectrally distinguish different types of amyloid aggregates. Herein, we report that heptamer formyl thiophene acetic acid (hFTAA) passes the blood-brain barrier after systemic administration and specifically binds to extracellular ß-amyloid deposits in the brain parenchyma (Aß plaques) and in the vasculature (cerebral ß-amyloid angiopathy) of ß-amyloid precursor protein transgenic APP23 mice. Moreover, peripheral application of hFTAA also stained intracellular lesions of hyperphosphorylated Tau protein in P301S Tau transgenic mice. Spectral profiling of all three amyloid types was acquired ex vivo using two-photon excitation. hFTAA revealed a distinct shift in its emission spectra when bound to Aß plaques versus Tau lesions. Furthermore, a spectral shift was observed for Aß plaques versus cerebral ß-amyloid angiopathy, indicating that different amyloid types and structural variances of a specific amyloid type can be distinguished. In conclusion, by adding spectral signatures to amyloid lesions, our results pave the way for a new area of in vivo amyloid imaging, allowing in vivo differentiation of amyloid (sub)types and monitoring changes of their structure/composition over time.