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Assessing comprehension difficulties requires the ability to assess cognitive load. Changes in cognitive load induced by comprehension difficulties could be detected with an adequate time resolution using different biofeedback measures (e.g., changes in the pupil diameter). However, identifying the Spatio-temporal sources of content comprehension difficulties (i.e., when, and where exactly the difficulty occurs in content regions) with a fine granularity is a big challenge that has not been explicitly addressed in the state-of-the-art. This paper proposes and evaluates an innovative approach named Intelligent BiofeedbackAugmented Content Comprehension (TellBack) to explicitly address this challenge. The goal is to autonomously identify regions of digital content that cause user's comprehension difficulty, opening the possibility to provide real-time comprehension support to users. TellBack is based on assessing the cognitive load associated with content comprehension through non-intrusive cheap biofeedback devices that acquire measures such as pupil response or Heart Rate Variability (HRV). To identify when exactly the difficulty in comprehension occurs, physiological manifestations of the Autonomic Nervous System (ANS) such as the pupil diameter variability and the modulation of HRV are exploited, whereas the fine spatial resolution (i.e., the region of content where the user is looking at) is provided by eye-tracking. The evaluation results of this approach show an accuracy of 83.00% ± 0.75 in classifying regions of content as difficult or not difficult using Support Vector Machine (SVM), and precision, recall, and micro F1-score of 0.89, 0.79, and 0.83, respectively. Results obtained with 4 other classifiers, namely Random Forest, k-nearest neighbor, Decision Tree, and Gaussian Naive Bayes, showed a slightly lower precision. TellBack outperforms the state-of-the-art in precision & recall by 23% and 17% respectively.

While dynamic ocular motor abnormalities (e.g., gaze-evoked nystagmus (GEN), low optokinetic nystagmus (OKN), pursuit and vestibulo-ocular reflex (VOR) gains, and dysmetric saccades) have been shown to be potential biomarkers in spinocerebellar ataxia type 3 (SCA3), the value of static abnormalities (e.g., convergent [esodeviation] and divergent strabismus [exodeviation]) is unknown. Moreover, studies on dynamic abnormalities in SCA3 usually do not take into account the existence of potential abduction-adduction asymmetries in patients with degenerative ataxia. Thirty-eight patients with genetically confirmed SCA3 (24 females; mean age ± SD, 49.8± 12.2 years) and 22 healthy controls (12 females, p = 0.589; mean age ± SD, 50.7± 12.5 years, p = 0.651) underwent clinical and video-oculographic assessment. A p value

Previous studies about the modulation of the vasculature by CO were performed exclusively in male or sexually immature animals. Understanding the sex differences regarding systemic drug processing and pharmacodynamics is an important feature for safety assessment of drug dosing and efficacy. In this work, we used CORM-A1 as source of CO to examine the effects of this gasotransmitter on brain perfusion and the sex-dependent differences. Dynamic contrast-enhanced imaging (DCE)-based analysis was used to characterize the properties of CO in the modulation of cerebral vasculature in vivo, in adult C57BL/6 healthy mice. Perfusion of the temporal muscle, maxillary vein and in hippocampus, cortex and striatum was analysed for 108 min following CORM-A1 administration of 3 or 5 mg/kg. Under control conditions, brain perfusion was lower in females when compared with males. Under CO treatment, females showed a surprisingly overall reduced perfusion compared with controls (F = 3.452, p = .0004), while no major alterations (or even the expected increase) were observed in males. Cortical structures were only modulated in females. A striking female-dominated vasoconstriction effect was observed in the hippocampus and striatum following administration of CO, in this mixed-sex cohort. As these two regions are implicated in episodic and procedural memory formation, CO may have a relevant impact in learning and memory.

The study of the physiological effects underlying brain response to transcranial magnetic stimulation is important to understand its impact on neurorehabilitation. We aim to analyze the impact of a transcranial magnetic stimulation protocol, the continuous theta burst (cTBS), on human neurophysiology, particularly on contralateral motor rhythms. cTBS was applied in 20 subjects over the primary motor cortex. We recorded brain electrical activity pre- and post-cTBS with electroencephalography both at rest and while performing motor tasks, to evaluate changes in brain oscillatory patterns such as mu and beta rhythms. Moreover, we measured motor-evoked potentials before and after cTBS to assess its impact on brain's excitability. On the hemisphere contralateral to the protocol, we did observe a significant increase in mu (p = 0.027) and beta (p = 0.006) rhythms from pre- to post-cTBS, at the beginning of arm elevation. The topology of action planning and motor execution suggests that cTBS produced an inhibitory effect that propagated to the contralateral hemisphere, thereby precluding the expected/desired excitation for therapy purposes. This novel approach provides support for the notion that this protocol induces inhibitory changes in contralateral motor rhythms, by decreasing desynchronization, contradicting the ipsilateral inhibition vs. contralateral disinhibition hypothesis. Our results have implications for personalized cTBS usage as a rehabilitation intervention, suggesting that an unexpected propagation of inhibition can occur.

Objectives: Transcranial magnetic stimulation, in particular continuous theta burst (cTBS), has been proposed for stroke rehabilitation, based on the concept that inhibition of the healthy hemisphere helps promote the recovery of the lesioned one. We aimed to study its effects on cortical excitability, oscillatory patterns, and motor function, the main aim being to identify potentially beneficial neurophysiological effects.Materials and Methods: We applied randomized real or placebo stimulation over the unaffected primary motor cortex of 10 subacute (7 ± 3 days) post-stroke patients. Neurophysiological measurements were performed using electroencephalography and electromyography. Motor function was assessed with the Wolf Motor Function Test. We performed a repeated measure study with the recordings taken pre-, post-cTBS, and at 3 months' follow-up.Results: We investigated changes in motor rhythms during arm elevation and thumb opposition tasks and found significant changes in beta power of the affected thumb's opposition, specifically after real cTBS. Our results are consistent with an excitatory response (increase in event-related desynchronization) in the sensorimotor cortical areas of the affected hemisphere, after stimulation. Neither peak-to-peak amplitude of motor-evoked potentials nor motor performance were significantly altered.Conclusions: Consistently with the theoretical prediction, this contralateral inhibitory stimulation paradigm changes neurophysiology, leading to a significant excitatory impact on the cortical oscillatory patterns of the contralateral hemisphere. These proof-of-concept results provide evidence for the potential role of continuous TBS in the neurorehabilitation of post-stroke patients. We suggest that these changes in ERS/ERD patterns should be further explored in future phase IIb/phase III clinical trials, in larger samples of poststroke patients.

An emergent research area in software engineering and software reliability is the use of wearable biosensors to monitor the cognitive state of software developers during software development tasks. The goal is to gather physiologic manifestations that can be linked to error-prone scenarios related to programmers’ cognitive states. In this paper we investigate whether electroencephalography (EEG) can be applied to accurately identify programmers’ cognitive load associated with the comprehension of code with different complexity levels. Therefore, a controlled experiment involving 26 programmers was carried. We found that features related to Theta, Alpha, and Beta brain waves have the highest discriminative power, allowing the identification of code lines and demanding higher mental effort. The EEG results reveal evidence of mental effort saturation as code complexity increases. Conversely, the classic software complexity metrics do not accurately represent the mental effort involved in code comprehension. Finally, EEG is proposed as a reference, in particular, the combination of EEG with eye tracking information allows for an accurate identification of code lines that correspond to peaks of cognitive load, providing a reference to help in the future evaluation of the space and time accuracy of programmers’ cognitive state monitored using wearable devices compatible with software development activities.

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3, the most common dominant spinocerebellar ataxia (SCA) worldwide, is caused by over-repetition of a CAG repeat in the ATXN3/MJD1 gene, which translates into a polyglutamine tract within the ataxin-3 protein. There is no treatment for this fatal disorder. Despite evidence of the safety and efficacy of mesenchymal stromal cells (MSCs) in delaying SCA disease progression in exploratory clinical trials, unanticipated regression of patients to the status prior to treatment makes the investigation of causes and solutions urgent and imperative. In the present study, we compared the efficacy of a single intracranial injection with repeated systemic MSC administration in alleviating the MJD phenotype of two strongly severe genetic rodent models. We found that a single MSC transplantation only produces transient effects, whereas periodic administration promotes sustained motor behavior and neuropathology alleviation, suggesting that MSC therapies should be re-designed to get sustained beneficial results in clinical practice. Furthermore, MSC promoted neuroprotection, increased the levels of GABA and glutamate, and decreased the levels of Myo-inositol, which correlated with motor improvements, indicating that these metabolites may serve as valid neurospectroscopic biomarkers of disease and treatment. This study makes important contributions to the design of new clinical approaches for MJD and other SCAs/polyglutamine disorders.

BACKGROUND Type-2 diabetes mellitus (T2DM) is a metabolic disorder with a broad range of complications in the brain that depend on the conditions that precede its onset, such as obesity and metabolic syndromes. It has been suggested that neurotransmitter and metabolic perturbations may emerge even before the early stages of T2DM and that high-caloric intake could adversely influence the brain in such states. Notwithstanding, evidence for neurochemical and structural alterations in these conditions are still sparse and controversial. PURPOSE To evaluate the influence of high-fat diet in the neurochemical profile and structural integrity of the rodent brain. STUDY TYPE Prospective. SUBJECTS Wistar rats (n = 12/group). FIELD STRENGTH/SEQUENCE A PRESS, ISIS, RARE, and EPI sequences were performed at 9.4T. ASSESSMENT Neurochemical and structural parameters were assessed by magnetic resonance spectroscopy, voxel-based morphometry, volumetry, and diffusion tensor imaging. STATISTICAL TESTS Measurements were compared through Student and Mann-Whitney tests. Pearson correlation was used to assess relationships between parameters. RESULTS Animals submitted to high-caloric intake gained weight (P = 0.003) and developed glucose intolerance (P

Methamphetamine (METH) abuse/misuse is a worldwide problem, and despite extensive characterization of its neurotoxicity over the last years, many questions remain unanswered. Recently, it was shown that METH compromises the blood-brain barrier (BBB) and causes a disturbance in the water homeostasis leading to brain edema. Importantly, water transport at BBB is regulated by water channels, aquaporins (AQPs), with AQP4 being expressed in astrocytic end-feet surrounding brain endothelium. Thus, the main goal of this work was to unravel the role of AQP4 under conditions of METH consumption. Our results show that METH (4× 10 mg/kg, 2 h apart, i.p.) interferes with AQP4 protein levels causing brain edema and BBB breakdown in both mice striatum and hippocampus, which culminated in locomotor and motivational impairment. Furthermore, these effects were prevented by pharmacological blockade of AQP4 with a specific inhibitor (TGN-020). Moreover, siRNA knockdown of this water channel protected astrocytes from METH-induced swelling and morphologic alterations. Herein, we unraveled AQP4 as a new therapeutic target to prevent the negative impact of METH.

Background The success of translational research depends on how well animal models mimic the pathophysiology of the human phenotype, and on the identification of disease mechanisms such as enhanced glycation. Methods Here, we studied cardiac MRI and metabolic phenotypes in human type 2 diabetes (N = 106; 55 patients+51 controls) and animal models with distinct levels of fat diet and end glycation products, to model the role of these factors in the cardiac phenotype. We included four groups of rats, designed to evaluate the role of lipid load and glucotoxicity in cardiac function and to correlate these with the cardiac phenotype observed in humans. We also aimed to assess into which extent phenotypes were related to specific risk factors. Results Stroke Volume (SV) and Peak Filling Rate (PFR) measures were similarly discriminative both in humans and animal models, particularly when enhanced glycation was present. Factorial analysis showed that reduction of multidimensionality into common main explanatory factors, in humans and animals, revealed components that equally explained the variance of cardiac phenotypes (87.62% and 83.75%, respectively). One of the components included, both in humans and animals, SV, PFR and peak ejection rate (PER). The other components included in both humans and animals are the following: ESV (end systolic volume), left ventricular mass (LVM) and ejection fraction (EF). These components were useful for between group discrimination. Conclusions We conclude that animal models of enhanced glycation and human type 2 diabetes share a striking similarity of cardiac phenotypic components and relation with metabolic changes, independently of fact content in the diet, which reinforces the role of glucose dysmetabolism in left ventricular dysfunction and provides a potentially useful approach for translational research in diabetes, in particular when testing new therapies early on during the natural history of this condition.

Recent studies have shown that programmers' cognitive load during typical code development activities can be assessed using wearable and low intrusive devices that capture peripheral physiological responses driven by the autonomic nervous system. In particular, measures such as heart rate variability (HRV) and pupillography can be acquired by nonintrusive devices and provide accurate indication of programmers' cognitive load and attention level in code related tasks, which are known elements of human error that potentially lead to software faults. This paper presents an experimental study designed to evaluate the possibility of using HRV and pupillography together with eye tracking to identify and annotate specific code lines (or even finer grain lexical tokens) of the program under development (or under inspection) with information on the cognitive load of the programmer while dealing with such lines of code. The experimental data is discussed in the paper to assess different alternatives for using code annotations representing programmers' cognitive load while producing or reading code. In particular, we propose the use of biofeedback code highlighting techniques to provide online programmer's warnings for potentially problematic code lines that may need a second look at (to remove possible bugs), and biofeedback-driven software testing to optimize testing effort, focusing the tests on code areas with higher bug probability

This study aims to investigate the relationship between structural changes in the retina and white matter in the brain, in early Alzheimer's disease (AD). Twenty-three healthy controls (mean age = 63.4±7.5 years) and seventeen AD patients (mean age = 66.5±6.6 years) were recruited for this study. By combining two imaging techniques-optical coherence tomography and diffusion tensor imaging (DTI)-the association between changes in the thickness of individual retinal layers and white matter dysfunction in early AD was assessed. Retinal layers were segmented, and thickness measurements were obtained for each layer. DTI images were analyzed with a quantitative data-driven approach to evaluating whole-brain diffusion metrics, using tract-based spatial statistics. Diffusion metrics, such as fractional anisotropy, are markers for white matter integrity. Multivariate and partial correlation analyses evaluating the association between individual retinal layers thickness and diffusion metrics were performed. We found that axial diffusivity, indexing axonal integrity, was significantly reduced in AD (p = 0.016, Cohen's d = 1.004) while in the retina, only a marginal trend for significance was found for the outer plexiform layer (p = 0.084, Cohen's d = 0.688). Furthermore, a positive association was found in the AD group between fractional anisotropy and the inner nuclear layer thickness (p < 0.05, r = 0.419, corrected for multiple comparisons by controlling family-wise error rate). Our findings suggest that axonal damage in the brain dominates early on in this condition and shows an association with retinal structural integrity already at initial stages of AD. These findings are consistent with an early axonal degeneration mechanism in AD.

This paper provides a study using Electroencephalography (EEG) to investigate the brain activity during code comprehension tasks. Three different code complexity levels according to five complexity metrics were considered. The use of EEG for this purpose is relevant, since the existing studies were mostly focused on neuroimaging techniques. Using Leave-One-Subject-Out cross-validation procedure for 30 subjects, it was found that the features related with the Gamma activity were the most common in all the folds. Regarding the brain regions, right parietal was the most frequent region contributing with more features. A Linear Discriminant Analysis Classifier for task classification, obtained a F-Measure of 92.71% for Code complexity easy, 52.25% for Code complexity intermediate and 53.13% for Code complexity advanced, revealing an evidence of mental effort saturation with the code complexity degree. This suggests that current code complexity metrics do not capture cognitive load and might not be the best approach to assess bug risk.

Our research explores a recent paradigm called Biofeedback Augmented Software Engineering (BASE) that introduces a strong new element in the software development process: the programmers' biofeedback. In this Practical Experience Report we present the results of an experiment to evaluate the possibility of using pupillography to gather biofeedback from the programmers. The idea is to use pupillography to get meta information about the programmers' cognitive and emotional states (stress, attention, mental effort level, cognitive overload,...) during code development to identify conditions that may precipitate programmers making bugs or bugs escaping human attention, and tag the corresponding code locations in the software under development to provide online warnings to the programmer or identify code snippets that will need more intensive testing. The experiments evaluate the use of pupillography as cognitive load predictor, compare the results with the mental effort perceived by programmers using NASATLX, and discuss different possibilities for the use of pupillography as biofeedback sensor in real software development scenarios.

This paper presents emergent experimental results showing that mental effort of programmers in code understanding tasks can be monitored through HRV (heart rate variability) using non-intrusive wearable devices. Results suggest that HRV is a good predictor for cognitive load when analyzing code and HRV results are consistent with the mental effort perceived by programmers using NASA-TLX. Furthermore, code complexity metrics do not correlate entirely with mental effort and do not seem a good indicator of the subjective perception of complexity felt by programmers. These first results are presented in the context of the project BASE-Biofeedback Augmented Software Engineering, which is briefly sketched, and proposes a radical neuroscience enabled approach to introduce biofeedback in software development.

Objective fMRI-based neurofeedback (NF) interventions represent the method of choice for the neuromodulation of localized brain areas. Although we have already validated an fMRI-NF protocol targeting the facial expressions processing network (FEPN), its dissemination is hampered by the economical and logistical constraints of fMRI-NF interventions, which may be however surpassed by transferring it to EEG setups, due to their low cost and portability. One of the major challenges of this procedure is then to reconstruct the BOLD-fMRI signal measured at the FEPN using only EEG signals. Because these types of approaches have been poorly explored so far, here we systematically investigated the extent at which the BOLD-fMRI signal recorded from the FEPN during a fMRI-NF protocol could be reconstructed from the simultaneously recorded EEG signal. Approach Several features from both scalp and source spaces (the latter estimated using continuous EEG source imaging) were extracted and used as predictors in a regression problem using random forests. Furthermore, three different approaches to deal with the hemodynamic delay of the BOLD signal were tested. The resulting models were compared with the only approach already proposed in the literature that uses spectral features and considers different time delays. Main results The combination of linear and non-linear features (particularly the largest Lyapunov exponent and entropy measures) projected into the source space, spatially filtered by independent component analysis (ICA) and convolved with multiple HRF functions peaking at different latencies, increases significantly the reconstruction accuracy (defined as the correlation between the measured and approximated BOLD signal) from 20% (direct comparison with the method used in the current literature) to 56%. Significance With this pipeline, a more accurate reconstruction of the BOLD signal can be obtained, which will positively impact the transfer of fMRI-based neurofeedback interventions to EEG setups, and more importantly, their dissemination and efficacy in modulating the activity of the desired brain areas.

Positron emission tomography (PET) neuroimaging with the Pittsburgh Compound_B (PiB) is widely used to assess amyloid plaque burden. Standard quantification approaches normalize PiB-PET by mean cerebellar gray matter uptake. Previous studies suggested similar pons and white-matter uptake in Alzheimer's disease (AD) and healthy controls (HC), but lack exhaustive comparison of normalization across the three regions, with data-driven diagnostic classification. We aimed to compare the impact of distinct reference regions in normalization, measured by data-driven statistical analysis, and correlation with cerebrospinal fluid (CSF) amyloid β (Aβ) species concentrations. 243 individuals with clinical diagnosis of AD, HC, mild cognitive impairment (MCI) and other dementias, from the Biomarkers for Alzheimer's/Parkinson's Disease (BIOMARKAPD) initiative were included. PiB-PET images and CSF concentrations of Aβ38, Aβ40 and Aβ42 were submitted to classification using support vector machines. Voxel-wise group differences and correlations between normalized PiB-PET images and CSF Aβ concentrations were calculated. Normalization by cerebellar gray matter and pons yielded identical classification accuracy of AD (accuracy-96%, sensitivity-96%, specificity-95%), and significantly higher than Aβ concentrations (best accuracy 91%). Normalization by the white-matter showed decreased extent of statistically significant multivoxel patterns and was the only method not outperforming CSF biomarkers, suggesting statistical inferiority. Aβ38 and Aβ40 correlated negatively with PiB-PET images normalized by the white-matter, corroborating previous observations of correlations with non-AD-specific subcortical changes in white-matter. In general, when using the pons as reference region, higher voxel-wise group differences and stronger correlation with Aβ42, the Aβ42/Aβ40 or Aβ42/Aβ38 ratios were found compared to normalization based on cerebellar gray matter., Highlights • Direct multivariate comparison of distinct reference regions in normalization of PET amyloid markers • Using the pons as ROI, higher voxel-wise group differences emerge • Using the pons as ROIs stronger correlation with Aβ42, the Aβ42/Aβ40 or Aβ42/Aβ38 ratios were found. • Evidence for statistical inferiority of CSF biomarkers • Aβ38 and Aβ40 correlated negatively with PiB-PET white-matter normalized images.

It remains an outstanding question whether gamma‐band oscillations reflect unitary cognitive processes within the same task. EEG/MEG studies do lack the resolution or coverage to address the highly debated question whether single gamma activity patterns are linked with multiple cognitive modules or alternatively each pattern associates with a specific cognitive module, within the same coherent perceptual task. One way to disentangle these issues would be to provide direct identification of their sources, by combining different techniques. Here, we directly examined these questions by performing simultaneous EEG/fMRI using an ambiguous perception paradigm requiring holistic integration. We found that distinct gamma frequency sub‐bands reflect different neural substrates and cognitive mechanisms when comparing object perception states vs. no categorical perception. A low gamma sub‐band (near 40 Hz) activity was tightly related to the decision making network, and in particular the anterior insula. A high gamma sub‐band (∼60 Hz) could be linked to early visual processing regions. The demonstration of a clear functional topography for distinct gamma sub‐bands within the same task shows that distinct gamma‐band modulations underlie sensory processing and perceptual decision mechanisms. Hum Brain Mapp 35:5219–5235, 2014. 2014 Wiley Periodicals, Inc.

We propose that understanding functional patterns of activity in mapped brain regions associated with code comprehension tasks and, more specifically, to the activity of finding bugs in traditional code inspections could reveal useful insights to improve software reliability and to improve the software development process in general. This includes helping to select the best professionals for the debugging effort, improving the conditions for code inspections, and identify new directions to follow for training code reviewers. This paper presents an interdisciplinary study to analyze the brain activity during code inspection tasks using functional magnetic resonance imaging (fMRI), which is a well-established tool in cognitive neuroscience research. We used several programs where realistic bugs representing the most frequent types of software faults found in the field were injected. The code inspectors involved in the research include programmers with different levels of expertise and experience in real code reviews. The goal is to understand brain activity patterns associated with code comprehension tasks and, more specifically, the brain activity when the code reviewer identifies a bug in the code ('eureka' moment), which can be a true positive or a false positive. Our results confirmed that brain areas associated with language processing and mathematics are highly active during code reviewing and shows that there are specific brain activity patterns that can be related to the decision-making moment of suspicion/bug detection. Importantly, the activity at the anterior insula region that we find to play a relevant role in the process of identifying software bugs is positively correlated to the precision of bug detection by the inspectors. This finding provides a new perspective on the role of this region on error awareness and monitoring and of its potential predictive value in predicting the quality of bug removing.

Saccadic intrusions are involuntary saccades that interrupt fixation and are usually conjugate between eyes.1 Although saccadic intrusions may be seen in healthy individuals, they also accompany certain neurologic disorders (usually manifesting higher frequency or amplitude) including cerebellar disease.2 We present a patient with spinocerebellar ataxia type 3 (SCA3) with an uncommon monocular/disconjugate alternating type of saccadic intrusion.

It has been hypothesized that neural synchrony underlies perceptual coherence. The hypothesis of loss of central perceptual coherence has been proposed to be at the origin of abnormal cognition in autism spectrum disorders and Williams syndrome, a neurodevelopmental disorder linked with autism, and a clearcut model for impaired central coherence. We took advantage of this model of impaired holistic processing to test the hypothesis that loss of neural synchrony plays a separable role in visual integration using EEG and a set of experimental tasks requiring coherent integration of local elements leading to 3-D face perception. A profound reorganization of brain activity was identified. Neural synchrony was reduced across stimulus conditions, and this was associated with increased amplitude modulation at 25–45 Hz. This combination of a dramatic loss of synchrony despite increased oscillatory activity is strong evidence that synchrony underlies central coherence. This is the first time, to our knowledge, that dissociation between amplitude and synchrony is reported in a human model of impaired perceptual coherence, suggesting that loss of phase coherence is more directly related to disruption of holistic perception.

An exciting area in cognitive neuroscience concerns the investigation of neural mechanisms underlying the emergence of visual perceptual representations. Multistable interpretations of the visual world are striking phenomena that may occur when the retinal image is compatible with multiple perceptual solutions. This occurs even when the physical pattern of retinal stimulation remains constant. Perceptual decision making is thus defined by the choice of an interpretation among a set of potentially ambiguous alternatives based on peripheral sensory evidence. We discuss current multimodal approaches to investigate the neural correlates of competition between multistable alternative neural representations and the generation of antagonistic perceptual interpretations.

The use of group-housed rodents in many fields of biomedical research imposes a need to identify individuals in a cage. Few studies have been designed to assess possible negative effects of identification methods of newborn mice on their development and wellbeing. In the present study, three different identification methods were applied to newborn C57BL/6J mice on postnatal day (pnd) 5 (toe clipping, toe tattoo ink puncture and subcutaneous implantation of a small transponder). All identification methods used proved to be effective for long-term marking of individual animals. Newborn mice showed the least reaction to toe clipping followed by toe tattoo ink puncture and transponder implantation was the most distressful individual identification procedure in newborn mice. Importantly, clipped toe tissue proved to be enough for genotyping purposes. No overall consistent differences in somatic and neurological reflex development during the postnatal period were shown as a result of the newborn individual identification procedures used. Further, none of the methods interfered significantly with the adult animals' general normal behaviour (e.g. ability to move, grasp, climb) and sensory-motor functions as assessed with a simplified SHIRPA battery of tests, as well as Rotarod and Elevated Plus Maze tests. Postmortem thymus and adrenal gland weights gave no indication of chronic stress as a consequence of the identification method. We conclude that toe clipping might even be advisable in newborn mice at a very young age, when genotyping is needed. Toe tattoo ink puncture is also a good identification method for newborn mice and transponder implantation should only be used in older newborns or applied at weaning., As this study was performed within the framework of the first author’s master thesis, thanks are due to Laboratory Animals Ltd for providing financial support.

Blood-brain barrier (BBB) disruption is a common feature in neurodegenerative diseases. However, BBB integrity has not been assessed in spinocerebellar ataxias (SCAs) such as Machado-Joseph disease/SCA type 3 (MJD/SCA3), a genetic disorder, triggered by polyglutamine-expanded ataxin-3. To investigate that, BBB integrity was evaluated in a transgenic mouse model of MJD and in human post-mortem brain tissues.Firstly, we investigated the BBB permeability in MJD mice by: i) assessing the extravasation of the Evans blue (EB) dye and blood-borne proteins (e.g fibrinogen) in the cerebellum by immunofluorescence, and ii) in vivo Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI). The presence of ataxin-3 aggregates in brain blood vessels and the levels of tight junction (TJ)-associated proteins were also explored by immunofluorescence and western blotting. Human brain samples were used to confirm BBB permeability by evaluating fibrinogen extravasation, co-localization of ataxin-3 aggregates with brain blood vessels and neuroinflammation.In the cerebellum of the mouse model of MJD, there was a 5-fold increase in EB accumulation when compared to age-matched controls. Moreover, vascular permeability displayed a 13-fold increase demonstrated by DCE-MRI. These results were validated by the 2-fold increase in fibrinogen extravasation in transgenic animals comparing to controls. Interestingly, mutant ataxin-3 aggregates were detected in cerebellar blood vessels of transgenic mice, accompanied by alterations of TJ-associated proteins in cerebellar endothelial cells, namely a 29% decrease in claudin-5 oligomers and a 10-fold increase in an occludin cleavage fragment. These results were validated in post-mortem brain samples from MJD patients as we detected fibrinogen extravasation across BBB, the presence of ataxin-3 aggregates in blood vessels and associated microgliosis.Altogether, our results prove BBB impairment in MJD/SCA3. These findings contribute for a better understanding of the disease mechanisms and opens the opportunity to treat MJD with medicinal products that in normal conditions would not cross the BBB.